Stress-responsive activator of p300 (strap) protein

ABSTRACT

The invention provides a protein which is a stress-responsive activator of the p300 protein, and nucleic acid sequences encoding the protein. The protein performs a key role in facilitating stress-responsive protein-protein interactions within the p300 co-activator complex. The STRAP protein facilitates the interaction of other proteins in the p300 complex, and is thus a target for assays for modulators of the complex.

This application is the US national phase of international applicationPCT/GB02/01349 filed 19 Mar. 2002, which designated the US.

FIELD OF INVENTION

This invention relates to the isolation and characterisation of a novelpolypeptide (Stress Responsive Activator of p53: STRAP) which is shownto interact with p300 and the p300 co-factor JMY in the p300co-activator complex. This complex is involved in the regulation of thetranscription of p53 target genes.

BACKGROUND OF INVENTION

The p53 protein is a stress-responsive transcription factor which isinduced by a variety of stimuli that act through mechanisms that alterp53 half-life (Ko and Prives, 1997; Levine, 1997). It is encoded by atumour suppressor gene that is frequently mutated in human cancer cells(Greenblat, 1994). In normal cells p53 exerts its function through thetargeted sequence-specific activation of a number of differentp53-responsive genes. These genes include waf1, bax, mdm2 and gadd45which encode proteins that give rise to the physiological consequencesof p53 activation, namely apoptosis or cell cycle arrest (Ko and Prives,1997). p53 alleles isolated from tumour cells frequently harbourmutations that disrupt p53 DNA binding activity (Cho et al., 1994),underscoring the importance of transcription-related functions inmediating the effects of p53 tumour suppression.

The pathways through which p53 activity is regulated have been subjectto intense study. The transcriptional activation domain of p53 istargeted by the MDM2 oncoprotein, which thereafter prevents p53 fromactivating transcription by hindering the interaction of p53 with thetranscription apparatus (Oliner et al., 1993; Lin et al., 1994). In thisrespect, MDM2 can override the physiological effects of p53 response (Wuet al., 1993), and it is consistent with this idea that mdm2 is oftenseen to be aberrantly expressed in human tumour cells (Piette et al.,1997). A further consequence of the interaction between MDM2 and p53 isa down-regulation in the level of p53 protein, which is mediated in partthrough a ubiquitin-dependent pathway and requires the MDM2 E3 ligase(Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997).

Because p53 inactivation is associated with many human cancers, researchhas been directed at restoring p53 function, in order to provide atherapy for these cancers. p53 may also make normal cells sensitive tostress and recently, research has also been directed at the temporaryinhibition of p53 (Komarova E. and Gudkov A. (1998) Seminars in CancerBiology 8(5) 389-400). This inhibition may be useful in ameliorating thep53 induced side effects of cancer therapies such as radio-andchemo-therapy, which include hair loss and damage to the lymphoid andhaematopoietic systems and the intestinal epithelia.

Adverse effects associated with the activation of P53 have also beendescribed in other conditions including injury associated cellularstress (e.g. burns), diseases associated with fever, local hypoxiaconditions associated with a deficient blood supply (e.g. stroke andischaemia) and cell aging (e.g. fibroblast senescence). Suppression ofp53 activity may therefore be useful in therapies related to theseconditions.

A considerable body of evidence supports a role for the p300/CBP familyof co-activators in p53-dependent transcription (Shikama et al., 1997).For example, p300/CBP proteins physically interact with the p53activation domain, and dominant-negative derivatives of p300/CBPproteins can block p53 activity (Avantaggiati et al., 1997; Gu et al.,1997; Lill et al., 1997; Lee et al., 1998). Moreover, phosphorylation ofthe p53 activation domain during the stress response is believed toantagonise the interaction with MDM2, and possibly stabilise theinteraction with p300/CBP proteins (Sheih et al., 1997; Giaccia andKastan, 1998; Chehab et al., 2000; Shieh et al., 2000). However,p300/CBP proteins function as integral components of largermulticomponent co-activator complexes, and a variety of co-factors thatmake up p300/CBP complexes have been identified (Shikama et al., 1997;Shiltz and Nakatani, 2000). Of considerable interest is the JMYco-factor, which is an integral component of the p300 co-activatorcomplex that augments the transcriptional activity of p53, and enhancesthe level of the p53 response (Shikama et al., 1999). No othercomponents of the p300 complex transcription have been characterisedwhich are involved in regulating p53.

SUMMARY OF INVENTION

The present inventors have isolated and characterised a new component ofthe p300 co-activator complex, which has been termed STRAP.

STRAP is composed almost entirely of a tandem array of tetratricopeptide(TPR) motifs (Lamb et al 1995) and can interact with distinct componentsof the p300 co-activator complex, such as p300 and the p300 co-factorJMY. STRAP augments the association between p300 and JMY and can inducethe p53 response, providing indication that STRAP plays a role inregulating the assembly of the p300 co-activator complex under stressconditions.

STRAP also undergoes a stress-responsive protein accumulation. Theability of STRAP to promote the interaction of co-factors within thep300 complex, combined with its response under cellular stress, endowSTRAP with a critical role in regulating the p53 response. Takentogether, these results identify a new and hitherto unexpected level ofcontrol, mediated at the point of stress-responsive co-activatorcontrol, in the cellular response to stress.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the primary amino acid sequence of murine STRAP (440 aminoacid residues, FIG. 1A, SEQ ID NO:1), human STRAP (440 amino acids, FIG.1B, SEQ ID NO:2), and the alignment of the two sequences (FIG. 1C).

FIG. 2 shows the cDNA nucleic acid sequence (SEQ ID NO:3) encoding theSTRAP amino acid sequence shown in FIG. 1A (SEQ ID NO:1).

FIG. 3 shows a diagrammatic summary of the distribution of the six TPRmotifs in STRAP (each TPR motif is indicated as I to VI andhighlighted).

FIG. 4 shows an alignment of the sequence from the six TPR motifs (SEQID NOs:4-9) in STRAP. The eight consensus residues derived from thealignment are shown below. The residue number is indicated at each endof the TPR motif. The consensus TPR motif is taken from Blatch andLässle (1999).

FIG. 5 shows the results of a two-hybrid assay in mammalian cells: theindicated expression vectors, either pVP16-JMY (0.5 μg) or pG4-Straptogether with the control (0.5 μg), pVP16 (0.5 μg) or G4 (0.1 μg)vectors were introduced into U2OS cells as indicated, together with thereporter pG5-luc (0.5 μg) and internal control pCMV-β-gal (1 μg). Thedata represent the relative activity of luciferase to β-galactosidaseand are the average values derived from two or more independentexperiments.

FIG. 6 shows the results of a two-hybrid assay performed in U2OS cellsas described in a) using the indicated vectors, namely pVP16-Strap (0.5μg) or pG4-p300 (0.5 μg). The data represent the average values derivedfrom two or more independent experiments.

FIG. 7 shows a summary of the binding domains in Strap, JMY and p300.The binding domains in JMY for p300, and in p300 for JMY, are taken fromShikama et al., 1999.

FIG. 8 shows the results of a two-hybrid assay performed in U2OS cellswith pG4-p300 (50 ng) and pVP-16-JMY (250 ng) in the presence ofincreasing amounts of pHA-Strap (0.5, 1.0, 3.0 and 5.0 μg), togetherwith the reporter pG5-luc (1 μg) and internal control pCMV-βgal (1 μg).The data represent the relative activity of luciferase toβ-galactosidase and are the average values derived from two or moredifferent readings.

FIG. 9 shows the indicated p53 reporter constructs, pWWP-luc, pBax-lucand pTG13 (1 μg) together with expression vectors for p53 (0.025 μg) andStrap (2 μg and 5 μg) were transfected into SAOS2 cells as indicated.The values shown represent the average of three separate readings andare the relative level of luciferase to β-galactosidase derived from theinternal control.

FIGS. 10 and 11 show the results of SAOS2 cells transfected withexpression vectors for wild-type p53 or p5322/23 (4 μg) in the presenceor absence of Strap (7 μg) together with pCMV CD20 (7 μg), as indicated.At 36 h after transfection, transfected cells were identified bystaining with anti-CD20 antibody, and DNA was stained with propidiumiodide, and the proportion of sub-G1, G1, S and G2/M phase cellsdetermined as described. The % change in the size of the sub-G1, G1, Sand G2/M population is shown. p53 caused about 37% of the transfectedcells to enter apoptosis, compared to 17% with the vector alone.

FIG. 12 shows the results of COS cells transfected with either pHA-Strap(5 μg), pCMV-JMY (5 μg) or pCMV-NAP2 (5 μg) and treated as describedeither with or without etoposide (200 or 400 nM). The levels ofexogenous Strap, JMY and NAP, and endogenous p53, were determined byimmunoblotting with the relevant antibodies, quantitated byphosphoimaging and thereafter presented graphically. The level ofprotein detected in the non-treated cell was given an arbitrary value of1.0. The symbols indicate: • p53, □ Strap, ▪ JMY and ∘ NAP.

FIG. 13 shows a two-hybrid assay performed in U2OS cells with pG4-p300(100 ng) and pVP16-JMY (1 μg) in the presence of pHA-Strap (2.5 μg) andetoposide (200 nM) as indicated, and the reporter pG5-luc (1 μg) andinternal control pCMV-βgal (1 μg). The data represent the relativeactivity of luciferase to β-galactosidase, and are the average valuesderived from two different readings.

FIG. 14 shows SAOS2 cells transfected with expression vectors for Strapor Strap8-123 (4 μg) and treated with etoposide (100 and 200 nM) asindicated. Exogenous p53 transcriptional activity was measured usingpBax-luc (1 μg), which was co-transfected together with expressionvectors for p53 (0.025 μg) and the internal control pCMV-βgal (1 μg).The values shown represent the average of three separate readings andare the relative level of luciferase.

FIG. 15 shows a possible model for role of Strap in the p53 response.Strap is induced by stress, and activates p300 co-activator function. Asa result, the p53 response is triggered to cause apoptosis or G1 arrest.Stress activated phosphokinases (including ATM, ATR and chk½) act uponp53 and MDM2 to alter p53 stability and enhance the interaction of p53with the p300 co-activator complex. Strap is induced by stress (?indicates that it is not known whether the same phosphokinases areinvolved) and augments the activity of the p300 co-activator complex byfacilitating co-factor interactions.

DETAILED DESCRIPTION OF INVENTION

According to one aspect of the present invention there is provided anisolated nucleic acid molecule encoding a polypeptide which includes theamino acid sequence shown in FIG. 1A (SEQ ID NO:1) or 1B (SEQ ID NO:2).For convenience, the sequences are referred to herein as the sequenceshown in FIG. 1, and reference to the sequence of FIG. 1 is intended toinclude both sequences unless specified explicitly to the contrary.

The coding sequence may be that shown included in FIG. 2 (SEQ ID NO:3)or it may be a mutant, variant, derivative or allele of the sequenceshown. A mutant, variant, derivative or allele may differ from thesequence shown by a change which is one or more of addition, insertion,deletion and substitution of one or more nucleotides of the sequenceshown. Changes to a nucleotide sequence may result in an amino acidchange at the protein level, or not, as determined by the genetic code.

Thus, nucleic acid according to the present invention may include asequence different from the sequence shown in FIG. 2 (SEQ ID NO:3) yetencode a polypeptide with the same amino acid sequence. The amino acidsequence shown in FIG. 1 (SEQ ID NO:1 or SEQ ID NO-2) consists of 440residues.

Alternatively, the encoded polypeptide may comprise an amino acidsequence which differs by one or more amino acid residues from the aminoacid sequence shown in FIG. 1 (SEQ ID NO:1 or SEQ ID NO:2). Nucleic acidencoding a polypeptide which is an amino acid sequence mutant, variant,derivative or allele of the sequence shown in FIG. 1 (SEQ ID NO:1 or SEQID NO:2) is further provided by the present invention. Such polypeptidesare discussed below. Nucleic acid encoding such a polypeptide may showat the nucleotide sequence and/or encoded amino acid level greater thanabout 60% homology with the coding sequence shown in FIG. 2 (SEQ IDNO:3) and/or the amino acid sequence shown in FIG. 1 (SEQ ID NO:1 or SEQID NO:2), greater than about 70% homology, greater than about 80%homology, greater than about 90% homology or greater than about 95%homology.

For amino acid “homology”, this may be understood to be similarity(according to the established principles of amino acid similarity, e.g.as determined using the algorithm GAP (Genetics Computer Group, Madison,Wis.) or identity. GAP uses the Needleman and Wunsch algorithm to aligntwo complete sequences that maximizes the number of matches andminimizes the number of gaps. Generally, the default parameters areused, with a gap creation penalty=12 and gap extension penalty=4. Use ofGAP may be preferred but other algorithms may be used, e.g. BLAST (whichuses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410),FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85:2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981)J. Mol. Biol. 147: 195-197), generally employing default parameters.

Another method for determining the best overall match between annucleotide or amino acid sequence of the present invention, or a portionthereof, and a query sequence is the use of the FASTDB computer programbased on the algorithm of Brutlag et al (Comp. App. Biosci., 6; 237-245(1990)). The program provides a global sequence alignment. The result ofsaid global sequence alignment is in percent identity. Suitableparameters used in a FASTDB search of a DNA sequence to calculatepercent identity are: Matrix=Unitary, k-tuple=4, Mismatch penalty=1,Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, GapPenalty=5, Gap Size Penalty=0.05, and Window Size=500 or query sequencelength in nucleotide bases, whichever is shorter. Suitable parameters tocalculate percent identity and similarity of an amino acid alignmentare: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty=0.05, and Window Size=500 or query sequence length in nucleotidebases, whichever is shorter.

Use of either of the terms “homology” and “homologous” herein does notimply any necessary evolutionary relationship between comparedsequences, in keeping for example with standard use of terms such as“homologous recombination” which merely requires that two nucleotidesequences are sufficiently similar to recombine under the appropriateconditions. Further discussion of polypeptides according to the presentinvention, which may be encoded by nucleic acid according to the presentinvention, is found below.

The present invention extends to nucleic acid that hybridizes with anyone or more of the specific sequences disclosed herein under stringentconditions. Such nucleic acid may include other animal, for example fish(such as the Zebra fish), worm (such as C. elegans) and particularlymammalian (e.g. rat or rabbit, sheep, goat, pig, or primate particularlyhuman) homologues of the STRAP gene. Such sequences may be obtained bymaking or obtaining cDNA libraries made from dividing cells or tissuesor genomic DNA libraries from other animal species, and probing suchlibraries with probes comprising all or part of a nucleic acid of theinvention under conditions of medium to high stringency.

Suitable conditions include, e.g. for detection of sequences that areabout 80-90% identical suitable conditions include hybridizationovernight at 42° C. in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextransulfate and a final wash at 55° C. in 0.1×SSC, 0.1% SDS. For detectionof sequences that are greater than about 90% identical, suitableconditions include hybridization overnight at 65° C. in 0.25M Na2HPO4,pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60° C. in0.1×SSC, 0.1% SDS.

A variant form of a nucleic acid molecule may contain one or moreinsertions, deletions, substitutions and/or additions of one or morenucleotides compared with the wild-type sequence (such as shown in FIG.2(SEQ ID NO:3)) which may or may not disrupt the gene function.Differences at the nucleic acid level are not necessarily reflected by adifference in the amino acid sequence of the encoded polypeptide.However, a mutation or other difference in a gene may result in aframe-shift or stop codon, which could seriously affect the nature ofthe polypeptide produced (if any), or a point mutation or grossmutational change to the encoded polypeptide, including insertion,deletion, substitution and/or addition of one or more amino acids orregions in the polypeptide. A mutation in a promoter sequence or otherregulatory region may prevent or reduce expression from the gene oraffect the processing or stability of the mRNA transcript. For instance,a sequence alteration may affect splicing of mRNA.

Generally, nucleic acid according to the present invention is providedas an isolate, in isolated and/or purified form, or free orsubstantially free of material with which it is naturally associated,such as free or substantially free of nucleic acid flanking the gene inthe human genome, except possibly one or more regulatory sequence(s) forexpression. Nucleic acid may be wholly or partially synthetic and mayinclude genomic DNA, cDNA or RNA. The coding sequence shown herein is aDNA sequence. Where nucleic acid according to the invention includesRNA, reference to the sequence shown should be construed as encompassingreference to the RNA equivalent, with U substituted for T.

Nucleic acid may be provided as part of a replicable vector, and alsoprovided by the present invention are a vector including nucleic acid asset out above, particularly any expression vector from which the encodedpolypeptide can be expressed under appropriate conditions, and a hostcell containing any such vector or nucleic acid. An expression vector inthis context is a nucleic acid molecule including nucleic acid encodinga polypeptide of interest and appropriate regulatory sequences forexpression of the polypeptide, in an in vitro expression system, e.g.reticulocyte lysate, or in vivo, e.g. in eukaryotic cells such as COS orCHO cells or in prokaryotic cells such as E. coli. This is discussedfurther below.

The nucleic acid sequence provided in accordance with the presentinvention is also useful in methods for identifying and/or obtainingnucleic acid of interest (and which may be according to the presentinvention) in a test sample, for example, homologues of the STRAPnucleotide sequence as described above.

A method of identifying and/or obtaining nucleic acid of interest mayinclude hybridisation of a probe having the sequence shown in FIG. 2(SEQ ID NO:3), or a complementary sequence, to target nucleic acid.

Hybridisation is generally followed by identification of successfulhybridisation and isolation of nucleic acid which has hybridised to theprobe, which may involve one or more steps of PCR. It will not usuallybe necessary to use a probe with the complete sequence shown in any ofthese figures. Shorter fragments, particularly fragments with a sequenceencoding the conserved TPR motifs may be used. Suitable sequences mayinclude sequences encoding one or more of the TPR motifs shown in FIG. 4(SEQ ID NOs:4-9).

Nucleic acids encoding or associated with the STRAP gene may also beused in methods of detecting the presence or absence of said gene in ahuman or non-human mammalian subject, said method comprising;

-   -   (a) bringing a sample of nucleic acid from said subject into        contact, under hybridizing conditions, with a polynucleotide of        the invention; and    -   (b) determining whether said polynucleotide has been able to        hybridize to a homologous sequence in said nucleic acid.

The method may be performed using a polynucleotide primer suitable foruse in a polymerase chain reaction (PCR), and the determining may beperformed in conjunction with a second primer using PCR such that aportion of the STRAP gene is amplified.

In one embodiment, the sample nucleic acid may be in the form of wholechromosomes, for example as a metaphase spread. The nucleic acid probeor primer of the invention may be labelled with a fluorescent label todetect the chromosomal location of a STRAP gene in the spread.

In some instances, the determining step may include determining thesequence of the STRAP gene, when present, in the nucleic acid sample. Asone alternative, restriction length fragment polymorphisms associatedwith the gene may be established and the assay performed with a samplewhich has been digested with a restriction enzyme. Another method ofdetermining is via PCR length polymorphisms, for example throughvariation in the sizes of introns. Other specific means of determininghybridization are well known and routine in the art and may also beused.

As well as determining the presence of polymorphisms or mutations in theSTRAP sequence, the probes may also be used to determine whether mRNAencoding the STRAP gene is present in a cell or tissue.

Nucleic acid according to the present invention is obtainable using oneor more oligonucleotide probes or primers designed to hybridise with oneor more fragments of the nucleic acid sequence shown in any of thefigures, particularly fragments of relatively rare sequence, based oncodon usage or statistical analysis. A primer designed to hybridise witha fragment of the nucleic acid sequence shown in any of the figures maybe used in conjunction with one or more oligonucleotides designed tohybridise to a sequence in a cloning vector within which target nucleicacid has been cloned, or in so-called “RACE” (rapid amplification ofcDNA ends) in which cDNA's in a library are ligated to anoligonucleotide linker and PCR is performed using a primer whichhybridises with a sequence shown and a primer which hybridises to theoligonucleotide linker.

Nucleic acid isolated and/or purified from one or more cells (e.g.human) or a nucleic acid library derived from nucleic acid isolatedand/or purified from cells (e.g. a cDNA library derived from mRNAisolated from the cells), may be probed under conditions for selectivehybridisation and/or subjected to a specific nucleic acid amplificationreaction such as the polymerase chain reaction (PCR) (reviewed forinstance in “PCR protocols; A Guide to Methods and Applications”, Eds.Innis et al, 1990, Academic Press, New York, Mullis et al, Cold SpringHarbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR technology,Stockton Press, NY, 1989, and Ehrlich et al, Science, 252:1643-1650,(1991)). PCR comprises steps of denaturation of template nucleic acid(if double-stranded), annealing of primer to target, and polymerization.The nucleic acid probed or used as template in the amplificationreaction may be genomic DNA, cDNA or RNA. Other specific nucleic acidamplification techniques include strand displacement activation, the Qβreplicase system, the repair chain reaction, the ligase chain reactionand ligation activated transcription. For convenience, and because it isgenerally preferred, the term PCR is used herein in contexts where othernucleic acid amplification techniques may be applied by those skilled inthe art. Unless the context requires otherwise, reference to PCR shouldbe taken to cover use of any suitable nucleic amplification reactionavailable in the art.

In the context of cloning, it may be necessary for one or more genefragments to be ligated to generate a full-length coding sequence. Also,where a full-length encoding nucleic acid molecule has not beenobtained, a smaller molecule representing part of the full molecule, maybe used to obtain full-length clones. Inserts may be prepared frompartial cDNA clones and used to screen cDNA libraries. The full-lengthclones isolated may be subcloned into expression vectors and activityassayed by transfection into suitable host cells, e.g. with a reporterplasmid.

A method may include hybridisation of one or more (e.g. two) probes orprimers to target nucleic acid. Where the nucleic acid isdouble-stranded DNA, hybridisation will generally be preceded bydenaturation to produce single-stranded DNA. The hybridisation may be aspart of a PCR procedure, or as part of a probing procedure not involvingPCR. An example procedure would be a combination of PCR and lowstringency hybridisation. A screening procedure, chosen from the manyavailable to those skilled in the art, is used to identify successfulhybridisation events and isolated hybridised nucleic acid.

Binding of a probe to target nucleic acid (e.g. DNA) may be measuredusing any of a variety of techniques at the disposal of those skilled inthe art. For instance, probes may be radioactively, fluorescently orenzymatically labelled. Other methods not employing labelling of probeinclude examination of restriction fragment length polymorphisms,amplification using PCR, RN'ase cleavage and allele specificoligonucleotide probing. Probing may employ the standard Southernblotting technique. For instance, DNA may be extracted from cells anddigested with different restriction enzymes. Restriction fragments maythen be separated by electrophoresis on an agarose gel, beforedenaturation and transfer to a nitrocellulose filter. Labelled probe maybe hybridised to the DNA fragments on the filter and binding determined.DNA for probing may be prepared from RNA preparations from cells.

Preliminary experiments may be performed by hybridising various probesunder low stringency conditions to Southern blots of DNA digested withrestriction enzymes. Suitable conditions would be achieved when a largenumber of hybridising fragments were obtained while the backgroundhybridisation was low. Using these conditions nucleic acid libraries,e.g. cDNA libraries representative of expressed sequences, may besearched. Those skilled in the art are well able to employ suitableconditions of the desired stringency for selective hybridisation, takinginto account factors such as oligonucleotide length and basecomposition, temperature and so on. On the basis of amino acid sequenceinformation, oligonucleotide probes or primers may be designed, takinginto account the degeneracy of the genetic code, and, where appropriate,codon usage of the organism from which the candidate nucleic acid isderived.

An oligonucleotide for use in nucleic acid amplification may have about10 or fewer codons (e.g. 6, 7 or 8), i.e. be about 30 or fewernucleotides in length (e.g. 18, 21 or 24). Generally specific primersare upwards of 14 nucleotides in length, but need not be than 18-20.Those skilled in the art are well versed in the design of primers foruse processes such as PCR. Various techniques for synthesizingoligonucleotide primers are well known in the art, includingphosphotriester and phosphodiester synthesis methods.

Preferred amino acid sequences suitable for use in the design of probesor PCR primers may include sequences conserved (completely,substantially or partly) encoding the TPR motifs.

A further aspect of the present invention provides an oligonucleotide orpolynucleotide fragment of the nucleotide sequence shown in any of thefigures herein providing nucleic acid according to the presentinvention, or a complementary sequence, in particular for use in amethod of obtaining and/or screening nucleic acid. A sequence may differfrom any of the sequences shown by addition, substitution, insertion ordeletion of one or more nucleotides, but preferably without abolition ofability to hybridise selectively with nucleic acid in accordance withthe present invention, that is wherein the degree of similarity of theoligonucleotide or polynucleotide with one of the sequences given issufficiently high.

In some preferred embodiments, oligonucleotides according to the presentinvention that are fragments of any of the sequences shown, are at leastabout 10 nucleotides in length, more preferably at least about 15nucleotides in length, more preferably at least about 20 nucleotides inlength. Such fragments themselves individually represent aspects of thepresent invention. Fragments and other oligonucleotides may be used asprimers or probes as discussed.

Further embodiments of oligonucleotides according to the presentinvention are anti-sense oligonucleotide sequences based on the nucleicacid sequences described herein. Anti-sense oligonucleotides may bedesigned to hybridise to the complementary sequence of nucleic acid,pre-mRNA or mature mRNA, interfering with the production of polypeptideencoded by a given DNA sequence (e.g. either native polypeptide or amutant form thereof), so that its expression is reduce or preventedaltogether. Anti-sense techniques may be used to target a codingsequence, a control sequence of a gene, e.g. in the 5′ flankingsequence, whereby the antisense oligonucleotides can interfere withcontrol sequences. Anti-sense oligonucleotides may be DNA or RNA and maybe of around 14-23 nucleotides, particularly around 15-18 nucleotides,in length. The construction of antisense sequences and their use isdescribed in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990), andCrooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, (1992).

Any of the sequences disclosed in the figures herein may be used toconstruct a probe for use in identification and isolation of a promoterfrom a genomic library containing a genomic STRAP gene. Techniques andconditions for such probing are well known in the art and are discussedelsewhere herein. To find minimal elements or motifs responsible forstress and/or developmental regulation, restriction enzyme or nucleasesmay be used to digest a nucleic acid molecule, followed by anappropriate assay (for example using a reporter gene such as luciferase)to determine the sequence required.

A further aspect of the present invention provides a nucleic acidmolecule as described herein operably linked to a promoter or otherregulatory sequence.

By “promoter” is meant a sequence of nucleotides from whichtranscription may be initiated of DNA operably linked downstream (i.e.in the 3′ direction on the sense strand of double-stranded DNA).

“Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional initiation regulation” of the promoter.

A further aspect of the present invention provides a polypeptide whichhas the amino acid sequence shown in FIG. 1 (SEQ ID NO:1 or SEQ IDNO:2), which may be in isolated and/or purified form, free orsubstantially free of material with which it is naturally associated,such as other polypeptides. If produced by expression in a prokaryoticcell) the polypeptide may be lacking in native glycosylation, e.g.unglycosylated. Polypeptides may of course be formulated with diluentsor adjuvants and still for practical purposes be isolated—for examplethe polypeptides will normally be mixed with gelatin or other carriersif used to coat microtitre plates for use in immunoassays. Polypeptidesmay be phosphorylated and/or acetylated.

Such a polypeptide is termed a STRAP polypeptide. This term alsoincludes amino acid sequence variants, alleles, derivatives and mutantsas well as active portions and fragments thereof.

The invention further provides active portions and fragments whichcomprises an epitope of said polypeptide. Unless otherwise specifiedbelow, such portions and fragments are also referred to as a polypeptideof the present invention.

Polypeptides which are amino acid sequence variants, alleles,derivatives or mutants are also provided by the present invention. Apolypeptide which is a variant, allele, derivative or mutant may have anamino acid sequence which differs from that given in a figure herein byone or more of addition, substitution, deletion and insertion of one ormore amino acids. Preferred such polypeptides have STRAP function, thatis to say have one or more of the following properties: immunologicalcross-reactivity with an antibody reactive the polypeptide for which thesequence is given in a figure herein; sharing an epitope with thepolypeptide for which the amino acid sequence is shown in a figureherein (as determined for example by immunological cross-reactivitybetween the two polypeptides); a biological activity which is inhibitedby an antibody raised against the polypeptide whose sequence is shown ina figure herein; ability to bind with p300 and/or JMY. Alteration ofsequence may change the nature and/or level of activity and/or stabilityof the STRAP polypeptide.

Polypeptides of the invention may be modified for example by theaddition of histidine residues to assist their purification or by theaddition of a signal sequence to promote their secretion from a cell.

A polypeptide which is an amino acid sequence variant, allele,derivative or mutant of the amino acid sequence shown in a figure hereinmay comprise an amino acid sequence which shares greater than about 35%sequence identity with the sequence shown, greater than about 40%,greater than about 50%, greater than about 60%, greater than about 70%,greater than about 80%, greater than about 90% or greater than about95%. The sequence may share greater than about 60% similarity, greaterthan about 70% similarity, greater than about 80% similarity or greaterthan about 90% similarity with the amino acid sequence shown in therelevant figure.

Amino acid similarity is generally defined with reference to thealgorithm GAP (Genetics Computer Group, Madison, Wis.) as noted above,or the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215:403-10. Similarity allows for “conservative variation”, i.e.substitution of one hydrophobic residue such as isoleucine, valine,leucine or methionine for another, or the substitution of one polarresidue for another, such as arginine for lysine, glutamic for asparticacid, or glutamine for asparagine. Particular amino acid sequencevariants may differ from that shown in a figure herein by insertion,addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-2020-30, 30-50, 50-100, 100-150, or more than 150 amino acids.

Sequence comparison may be made over the full-length of the relevantsequence shown herein, or may more preferably be over a contiguoussequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133,167, 200, 233, 267, 300, 333, 400, or more amino acids, compared withthe relevant amino acid sequence as the case may be.

Preferred such polypeptides include those which are encoded by the STRAPgene of other mammals, particularly primates and most particularly man,as well as fragments of such polypeptides, such fragments being those asdefined above. The primary sequence of the STRAP protein will besubstantially similar to that of FIG. 1 (SEQ ID NO:1 or SEQ ID NO:2) andmay be determined by routine techniques available to those of skill inthe art. In essence, such techniques comprise using polynucleotides ofthe present invention as probes to recover and to determine the sequenceof the STRAP gene in other species. Human STRAP is shown in FIG. 1B (SEQID NO:2), and was obtained in the light of the present invention byanalysis of publicly available sequence databases. The databases do notidentify that this protein as such, nor identify the function that isdisclosed herein.

A wide variety of techniques are available for this, for example PCRamplification and cloning of the gene using a suitable source of mRNA(e.g. from an embryo or an actively dividing differentiated or tumourcell), or by methods comprising obtaining a cDNA library from themammal, e.g a cDNA library from one of the above-mentioned sources,probing said library with a polynucleotide of the invention understringent conditions, and recovering a cDNA encoding all or part of theSTRAP protein of that mammal. Where a partial cDNA is obtained, the fulllength coding sequence may be determined by primer extension techniques.

The present invention also includes peptides which include or consist offragments of a polypeptide of the invention.

The present inventors have also identified regions in the JMY and p300sequence which interact with STRAP. A peptide consisting of such aregion and nucleic acid encoding such a peptide are further aspects ofthe present invention.

Regions of JMY which interact with STRAP include residues 683 to 983 andregions of p300 which interact with STRAP include residues 1 to 595 andresidues 1572 to 1921. The sequence of JMY is available as GenBankaccession no. AAF 17555 and the sequence of p300 available as GenBankaccession number XP010013.

“p300” refers to a family member of the p300/CBP family of co-activatorswhich have histone acetyltransferase activity p300 is described forexample by Eckner et al, 1994 and CBP by Bannister and Kouzarides, 1996.For the purposes of the present invention, reference to “p300” or “p300polypeptide” refers to human allelic and synthetic variants of p300 orCBP, and to other mammalian variants and allelic and synthetic variantsthereof, as well as fragments of said human and mammalian forms of p300or CBP. Synthetic variants include those which have at least 80%,preferably at least 90%, homology to p300. More preferably such variantscorrespond to the sequence of p300 but have one or more, e.g. from 1 to10, such as from 1 to 5, substitutions, deletions or insertions of aminoacids. Fragments of p300 and its variants are preferably at least 20,more preferably at least 50 and most preferably at least 200 amino acidsin size. The p300 molecule will however retain the ability to physicallyassociate in vivo with STRAP.

Preferably, the p300 used in assays of the present invention will alsoretain the ability to interact with the tumour suppressor molecule p53,as described in the accompanying examples and by Lill et al, 1997.

For the purposes of the present invention, the precise form andstructure of a p300 protein or fragment thereof may be varied by thoseof skill in the art, having regard to the particular assay format to beused.

“JMY” refers to any family member of the JMY family of co-activatorswhich bind the p300/CBP co-activator complex and are disclosed inPCT/GB98/03152. For the purposes of the present invention, reference to“JMY” or “JMY polypeptide” refers to human allelic and syntheticvariants of JMY, and to other mammalian variants and allelic andsynthetic variants thereof, as well as fragments of said human andmammalian forms of JMY. Synthetic variants include those which have atleast 80%, preferably at least 90%, homology to JMY. More preferablysuch variants correspond to the sequence of JMY but have one or more,e.g. from 1 to 10, such as from 1 to 5, substitutions, deletions orinsertions of amino acids. Fragments of JMY and its variants arepreferably at least 20, more preferably at least 50 and most preferablyat least 200 amino acids in size. The JMY molecule will however retainthe ability to physically associate in vivo with STRAP and/or p300.

For the purposes of the present invention, the precise form andstructure of a JMY protein or fragment thereof may be varied by those ofskill in the art, having regard to the particular assay format to beused.

“p53” refers to the tumour suppressor gene or its encoded amino acidsequence of as reported, for example, by Matlashewski et al (EMBO J. 13;3257-62, 1984) or Lamb and Crawford (Mol. Cell. Biol. 5; 1379-85, 1986).These sequences are available on Genbank. Wild-type human p53 proteinincludes a proline/arginine polymorphism at amino acid 72, reflecting acorresponding polymorphism in the gene.

The skilled person can use the techniques described herein and otherswell known in the art to produce large amounts of peptides, for instanceby expression from encoding nucleic acid.

Peptides can also be generated wholly or partly by chemical synthesis.The compounds of the present invention can be readily prepared accordingto well-established, standard liquid or, preferably, solid-phase peptidesynthesis methods, general descriptions of which are broadly available(see, for example, in J. M. Stewart and J. D. Young, Solid Phase PeptideSynthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984),in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis,Springer Verlag, New York (1984); and Applied Biosystems 430A UsersManual, ABI Inc., Foster City, Calif.).

The present invention also includes active portions, fragments,derivatives and functional mimetics of the polypeptides of theinvention. An “active portion” of a polypeptide means a peptide which isless than said full length polypeptide, but which retains a biologicalactivity, such as binding to p300 and/or JMY. Thus an active portion ofthe STRAP polypeptide may include amino acids 1 to 123 or amino acids123 to 205 which bind JMY and/or may include amino acids 206 to 440,which bind p300. Such an active fragment may be included as part of afusion protein, e.g. including a binding portion for a different ligandwhich may confer on the molecule a different binding specificity.

Active portions may also include those which are phosphorylated and/oracetylated, particularly in a cell-cycle specific manner.

A “fragment” of a polypeptide generally means a stretch of amino acidresidues of at least about five contiguous amino acids, often at leastabout seven contiguous amino acids, typically at least about ninecontiguous amino acids, more preferably at least about 13 contiguousamino acids, and, more preferably, at least about 20 to 30 or morecontiguous amino acids. Fragments of the STRAP polypeptide sequence mayinclude antigenic determinants or epitopes useful for raising antibodiesto a portion of the amino acid sequence. Alanine scans are commonly usedto find and refine peptide motifs within polypeptides, this involvingthe systematic replacement of each residue in turn with the amino acidalanine, followed by an assessment of biological activity.

Preferred fragments of STRAP include those which contain any of thefollowing amino acid sequences shown in FIG. 1 (SEQ ID NO: 1 or SEQ IDNO:2): residues 1 to 123, residues 122 to 205, residues 206 to 440,which may be used for instance in raising or isolating antibodies.Variant and derivative peptides, peptides which have an amino acidsequence which differs from one of these sequences by way of addition,insertion, deletion or substitution of one or more amino acids are alsoprovided by the present invention, generally with the proviso that thevariant or derivative peptide is bound by an antibody or other specificbinding member which binds one of the peptides whose sequence is shown.A peptide which is a variant or derivative of one of the shown peptidesmay compete with the shown peptide for binding to a specific bindingmember, such as an antibody or antigen-binding fragment thereof.

Where additional amino acids are included in a peptide, these may beheterologous or foreign to the polypeptide of the invention, and thepeptide may be about 20, 25, 30 or 35 amino acids in length. A peptideaccording to this aspect may be included within a larger fusion protein,particularly where the peptide is fused to a non-STRAP (i.e.heterologous or foreign) sequence, such as a polypeptide or proteindomain.

A “derivative” of a polypeptide or a fragment thereof may include apolypeptide modified by varying the amino acid sequence of the protein,e.g. by manipulation of the nucleic acid encoding the protein or byaltering the protein itself. Such derivatives of the natural amino acidsequence may involve one or more of insertion, addition, deletion orsubstitution of one or more amino acids, which may be withoutfundamentally altering the qualitative nature of biological activity ofthe wild type polypeptide.

Also encompassed within the scope of the present invention arefunctional mimetics of active fragments of the STRAP polypeptidesprovided (including alleles, mutants, derivatives and variants). Theterm “functional mimetic” means a substance which may not contain anactive portion of the relevant amino acid sequence, and probably is nota peptide at all, but which retains in qualitative terms a biologicalactivity of natural STRAP polypeptide. The design and screening ofcandidate mimetics is described in detail below.

A polypeptide according to the present invention may be isolated and/orpurified (e.g. using an antibody) for instance after production byexpression from encoding nucleic acid (for which see below). Thus, apolypeptide may be provided free or substantially free from contaminantswith which it is naturally associated (if it is a naturally-occurringpolypeptide). A polypeptide may be provided free or substantially freeof other polypeptides.

Polypeptides according to the present invention may be generated whollyor partly by chemical synthesis. The isolated and/or purifiedpolypeptide may be used in formulation of a composition, which mayinclude at least one additional component, for example a pharmaceuticalcomposition including a pharmaceutically acceptable excipient, vehicleor carrier. A composition including a polypeptide according to theinvention may be used in prophylactic and/or therapeutic treatment asdiscussed below.

A polypeptide, peptide, allele, mutant, derivative or variant accordingto the present invention may be used as an immunogen or otherwise inobtaining specific antibodies. Antibodies are useful in purification andother manipulation of polypeptides and peptides and therapeuticcontexts. This is discussed further below.

A polypeptide of the invention may be labelled with a revealing label.The revealing label may be any suitable label which allows thepolypeptide to be detected. Suitable labels include radioisotopes, e.g.125I, enzymes, antibodies, polynucleotides and linkers such as biotin.Labelled polypeptides of the invention may be used in diagnosticprocedures such as immunoassays in order to determine the amount of apolypeptide of the invention in a sample. Polypeptides or labelledpolypeptides of the invention may also be used in serological or cellmediated immune assays for the detection of immune reactivity to saidpolypeptides in animals and humans using standard protocols.

A polypeptide or labelled polypeptide of the invention or fragmentthereof may also be fixed to a solid phase, for example the surface ofan immunoassay well or dipstick.

Such labelled and/or immobilized polypeptides may be packaged into kitsin a suitable container along with suitable reagents, controls,instructions and the like.

Such polypeptides and kits may be used in methods of detection ofantibodies to such polypeptides present in a sample or active portionsor fragments thereof by immunoassay.

Immunoassay methods are well known in the art and will generallycomprise:

-   -   (a) providing a polypeptide comprising an epitope bindable by an        antibody against said protein;    -   (b) incubating a biological sample with said polypeptide under        conditions which allow for the formation of an antibody-antigen        complex; and    -   (c) determining whether antibody-antigen complex comprising said        polypeptide is formed.

A polypeptide according to the present invention may be used inscreening for molecules which affect or modulate its activity orfunction, e.g its interactions with p300 co-activator complex and/or itseffect on p53 activity. Such molecules may interact with the N terminalregion (residues 1 to 123), a region between amino acids 124 to 205 or aC terminal (residues 206 to 440) region of STRAP or with one or moreregions of JMY and/or p300 which bind to STRAP, and may be useful in atherapeutic (including prophylactic) context.

It is well known that pharmaceutical research leading to theidentification of a new drug may involve the screening of very largenumbers of candidate substances, both before and even after a leadcompound has been found. This is one factor which makes pharmaceuticalresearch very expensive and time-consuming. Means for assisting in thescreening process can have considerable commercial importance andutility. Such means for screening for substances potentially useful inmodulating (i.e. activating or reducing) the activity of p53 andtreating or preventing p53 induced apoptosis are provided bypolypeptides according to the present invention.

Substances identified as modulators of the interactions described hereinare extremely useful in the modulation of a range of stress related p53activities since they provide basis for design and investigation oftherapeutics for in vivo use. Furthermore, they may be useful in any ofa number of conditions in which p53 activity is undesirable, includingcancer-therapy genotoxicity, p53 dependent neuronal death in the centralnervous system (i.e. brain or spinal cord injury), preservation oftissues or organs prior to transplant, preparation of host for bonemarrow transplant, reducing neuronal damage during seizures, andsuppression of cell aging. As noted elsewhere, STRAP and fragmentsthereof may also be useful in combating any of these conditions anddisorders.

In various further aspects the present invention relates to screeningand assay methods and means, and substances identified thereby.

The identification of the polypeptide expressed by the STRAP geneenables assays to be developed to identify further cellular proteinswith which the polypeptide is associated, in addition to p300 and JMY.For example, polypeptides of the present invention may be required in aregulatory pathway in which their function is to interact with otherfactors which in turn promote or maintain essential cellular functionsassociated with cell cycle control. The polypeptides of the presentinvention may be used in two-hybrid assays as described below todetermine cellular factors with which they become associated.

Assay methods may therefore be for substances or agents which interactwith or bind a polypeptide of the invention and/or modulate one or moreof its activities.

Further aspects of the present invention provide the use of a STRAPpolypeptide or peptide (particularly a fragment of a polypeptide of theinvention as disclosed), and/or encoding nucleic acid therefor, inscreening or searching for and/or obtaining/identifying a substance,e.g. peptide or chemical compound, which interacts and/or binds with theSTRAP polypeptide or peptide and/or interferes with the interactionbetween STRAP and JMY and/or STRAP and p300 and which is therefore acandidate modulator of the function or activity of the p300 co-factorcomplex. Such a substance may be useful in modulating the activity ofp53, for example by means of modulating the half life of p53 and/ormodulating the activity of target genes which execute the p53 stressresponse.

For instance, a method according to one aspect of the invention includesproviding a polypeptide or peptide of the invention and bringing it intocontact with a substance, which contact may result in binding betweenthe polypeptide or peptide and the substance. Binding may be determinedby any of a number of techniques available in the art, both qualitativeand quantitative.

A method of screening for a substance which modulates the bindingactivity of a STRAP polypeptide may include contacting one or more testsubstances with the STRAP polypeptide in a suitable reaction medium,testing the binding activity of the treated polypeptide and comparingthat activity with the binding activity of the STRAP polypeptide incomparable reaction medium untreated with the test substance orsubstances. A difference in binding activity between the treated anduntreated polypeptides is indicative of a modulating effect of therelevant test substance or substances.

Corresponding aspects of the present invention relate to methods ofscreening for a substance which modulates the binding activity of apolypeptide consisting of residues 683 to 983 of JMY or a polypeptideconsisting of residues 1 to 595 or 1572 to 1921 of p300.

In various aspects the present invention is concerned with provision ofassays for substances which inhibit interaction between a polypeptide ofthe invention and one or more of JMY and p300.

One aspect of the present invention provides an assay method whichincludes:

-   -   (i) bringing into contact a STRAP polypeptide according to the        invention and a putative binding molecule or other test        substance; and    -   (ii) determining interaction or binding between the STRAP        polypeptide and the test substance.

A substance which interacts with a STRAP polypeptide or peptide of theinvention may be isolated and/or purified, manufactured and/or used tomodulate its activity as discussed.

A further aspect of the present invention provides an assay method forscreening for a substance which modulates the binding of JMY and STRAP,including:

-   -   (i) bringing a STRAP polypeptide into contact with a JMY        polypeptide in the presence of one or more test substances; and    -   (ii) determining the binding of the STRAP polypeptide to the JMY        polypeptide.

The STRAP polypeptide may be brought into contact with the JMYpolypeptide in the presence of a p300 polypeptide.

Another aspect of the present invention provides an assay method forscreening for a substance which modulates the binding of p300 and STRAP,including:

-   -   (i) bringing a STRAP polypeptide into contact with a p300        polypeptide in the presence of one or more test substances; and    -   (ii) determining the binding of the STRAP polypeptide to the        p300 polypeptide.

The STRAP polypeptide may be brought into contact with the p300polypeptide in the presence of a JMY polypeptide.

Another aspect of the present invention provides an assay method forscreening for a substance which modulates the binding of JMY, p300 andSTRAP, including:

-   -   (i) bringing a STRAP polypeptide into contact with a p300        polypeptide and a JMY polypeptide in the presence of one or more        test substances; and    -   (ii) determining the binding of the STRAP polypeptide to the        p300 polypeptide and the JMY polypeptide.

An assay may be carried out under conditions in which in the absence ofthe test substance being an inhibitor, the STRAP polypeptide binds tothe JMY or p300 polypeptide.

In assays of the present invention, the binding of a JMY and/or p300polypeptide to a STRAP polypeptide may be determined in the presence andabsence of the test substance. A difference in binding activity in thepresence of test substance is indicative of a modulating effect of therelevant test substance or substances.

An assay method may comprise determining the p53 stress response in thepresence and/or absence of said test substance as described herein.

As mentioned above, it is not necessary to use the entire proteins forassays of the invention which test for binding between two molecules.Fragments may be generated and used in any suitable way known to thoseof skill in the art. Suitable ways of generating fragments include, butare not limited to, recombinant expression of a fragment from encodingDNA. Such fragments may be generated by taking encoding DNA, identifyingsuitable restriction enzyme recognition sites either side of the portionto be expressed, and cutting out said portion from the DNA. The portionmay then be operably linked to a suitable promoter in a standardcommercially available expression system. Another recombinant approachis to amplify the relevant portion of the DNA with suitable PCR primers.Small fragments (e.g. up to about 20 or 30 amino acids) may also begenerated using peptide synthesis methods which are well known in theart.

The precise format of the assay of the invention may be varied by thoseof skill in the art using routine skill and knowledge. For example, theinteraction between the polypeptides may be studied in vitro bylabelling one with a detectable label and bringing it into contact withthe other which has been immobilised on a solid support. Suitabledetectable labels include 35S-methionine which may be incorporated intorecombinantly produced peptides and polypeptides. Recombinantly producedpeptides and polypeptides may also be expressed as a fusion proteincontaining an epitope which can be labelled with an antibody.

Fusion proteins may be generated that incorporate six histidine residuesat either the N-terminus or C-terminus of the recombinant protein. Sucha histidine tag may be used for purification of the protein by usingcommercially available columns which contain a metal ion, either nickelor cobalt (Clontech, Palo Alto, Calif., USA). These tags also serve fordetecting the protein using commercially available monoclonal antibodiesdirected against the six histidine residues (Clontech, Palo Alto,Calif., USA).

The protein which is immobilized on a solid support may be immobilizedusing an antibody against that protein bound to a solid support or viaother technologies which are known per se. A preferred in vitrointeraction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described above atest compound can be assayed by determining its ability to diminish theamount of labelled peptide or polypeptide which binds to the immobilizedGST-fusion polypeptide. This may be determined by fractionating theglutathione-agarose beads by SDS-polyacrylamide gel electrophoresis.Alternatively, the beads may be rinsed to remove unbound protein and theamount of protein which has bound can be determined by counting theamount of label present in, for example, a suitable scintillationcounter.

In an alternative mode, one of STRAP polypeptide and p300 or JMYpolypeptide may be labelled with a fluorescent donor moiety and theother labelled with an acceptor which is capable of reducing theemission from the donor. This allows an assay according to the inventionto be conducted by fluorescence resonance energy transfer (FRET). Inthis mode, the fluorescence signal of the donor will be altered whenSTRAP and p300 or JMY interact. The presence of a candidate modulatorcompound which modulates the interaction will increase the amount ofunaltered fluorescence signal of the donor.

FRET is a technique known per se in the art and thus the precise donorand acceptor molecules and the means by which they are linked to STRAPand p300 or JMY may be accomplished by reference to the literature.

Suitable fluorescent donor moieties are those capable of transferringfluorogenic energy to another fluorogenic molecule or part of a compoundand include, but are not limited to, coumarins and related dyes such asfluoresceins, rhodols and rhodamines, resorufins, cyanine dyes, bimanes,acridines, isoindoles, dansyl dyes, aminophthalic hydrazines such asluminol and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, and europium and terbium complexes and relatedcompounds.

Suitable acceptors include, but are not limited to, coumarins andrelated fluorophores, xanthenes such as fluoresceins, rhodols andrhodamines, resorufins, cyanines, difluoroboradiazaindacenes, andphthalocyanines.

A preferred donor is fluorescein and preferred acceptors includerhodamine and carbocyanine. The isothiocyanate derivatives of thesefluorescein and rhodamine, available from Aldrich Chemical Company Ltd,Gillingham, Dorset, UK, may be used to label STRAP or JMY or p300/CBP.For attachment of carbocyanine, see for example Guo et al, J. Biol.Chem., 270; 27562-8, 1995.

Another assay format is dissociation enhanced lanthanide fluorescentimmunoassay (DELFIA) (Ogata et al, 1992). This is a solid phase basedsystem for measuring the interaction of two macromolecules. Typicallyone molecule (either STRAP or JMY or p300) is immobilised to the surfaceof a multi well plate and the other molecule is added in solution tothis. Detection of the bound partner is achieved by using a labelconsisting of a chelate of a rare earth metal. This label can bedirectly attached to the interacting molecule or may be introduced tothe complex via an antibody to the molecule or to the molecules epitopetag. Alternatively, the molecule may be attached to biotin and astreptavidin-rare earth chelate used as the label. The rare earth usedin the label may be europium, samarium, terbium or dysprosium. Afterwashing to remove unbound label, a detergent containing low pH buffer isadded to dissociate the rare earth metal from the chelate. The highlyfluorescent metal ions are then quantitated by time resolvedfluorimetry. A number of labelled reagents are commercially availablefor this technique, including streptavidin, antibodies againstglutathione-S-transferase and against hexahistidine.

An assay according to the present invention may also take the form of anin vivo assay. The in vivo assay may be performed in a cell line such asa yeast strain in which the relevant polypeptides or peptides areexpressed from one or more vectors introduced into the cell.

In vivo assays may also take the form of two-hybrid assays wherein STRAPand p300 or JMY are expressed as fusion proteins, one being a fusionprotein comprising a DNA binding domain (DBD), such as the yeast GAL4binding domain, and the other being a fusion protein comprising anactivation domain, such as that from GAL4 or VP16. In such a case thehost cell (which again may be bacterial, yeast, insect or mammalian,particularly yeast or mammalian) will carry a reporter gene constructwith a promoter comprising a DNA binding elements compatible with theDBD.

STRAP and JMY or p300 and the reporter gene, may be introduced into thecell and expressed transiently or stably.

Alternatively, assays of the invention may be conducted by utilizing theability of a STRAP-p300 complex (including JMY) to mediate theactivation of a reporter gene or to induce a cellular response in acell, particularly apoptosis. For example, a number of transcriptionfactors, including the glucocorticoid receptor (GR) and E2F-1, are knownto be regulated by p300/CBP, as is p53. We have found that theregulation of such factors is enhanced by STRAP. Further, we have foundthat p53-mediated apoptosis is enhanced by the presence of STRAP.

Thus assays of the invention include an assay for a modulator ofSTRAP-p300 complex formation which comprises:

-   -   a) providing STRAP, p300 and JMY together with a regulatory        factor which is a target for p300, in the presence of a putative        modulator and a reporter gene which comprises a target promoter        for said regulatory factor; and    -   b) measuring the modulation of transcription of the reporter        gene caused by the presence of said modulator.

The regulatory factor includes GR for which suitable promoters includepromoters which contain a GRE such as c-myc and the MMLV LTR; E2F-1 forwhich suitable promoters include cyclin A, cyclin E, tyrosine aminotransferase and the E2F-1 gene promoter; p53 for which suitablepromoters include Bax, Waf1/Cip, Gadd45 and cyclin G; oestrogen receptor(ER) for which suitable promoters include progesterone receptor andPS-2; and other nuclear receptors and promoters containing recognitionelements of this type. Suitable reporter genes operably linked to thepromoter include chloramphenicol acetyl transferase, luciferase, greenfluorescent protein and β-galactosidase. In the case of ER, a the 13base palindromic estrogen response element (ERE) may be included in thepromoter of a reporter construct to provide a suitable reporter gene. Inan alternative embodiment, the assay may be conducted in a cell lackingwild-type p53 and which undergoes apoptosis in the presence of p53. Suchcells include SAOS-2 cells.

In this format the assay will be conducted by supplying to the cellexpression vector(s) encoding STRAP, JMY, p300/CBP and wild type p53,treating said cells with a putative modulator and measuring the effectof the modulator on apoptosis of the cells. Apoptosis may also bemeasured in an analogous manner in cell lines with wild type p53 whereinapoptosis is enhanced by the presence of, for example, excess STRAP.

Assays will be run with suitable controls routine to those of skill inthe art.

Accordingly, another aspect of the present invention is a substanceobtainable using an assay method as described herein.

Such a substance may include polypeptide, antibody, peptide, nucleicacid molecule, small molecule or other pharmaceutically useful compound.In some embodiments of this aspect of the invention, a polypeptide hasless than 900 residues and/or does not include the full length JMY andp300 sequences.

Combinatorial library technology (Schultz, J S (1996) Biotechnol. Prog.12:729-743) provides an efficient way of testing a potentially vastnumber of different substances for ability to modulate activity of apolypeptide. Prior to, or as well as, being screened for modulation ofactivity, test substances may be screened for ability to interact withthe polypeptide, e.g. in a yeast two-hybrid system (which requires thatboth the polypeptide and the test substance can be expressed in yeastfrom encoding nucleic acid). This may be used as a coarse screen priorto testing a substance for actual ability to modulate activity of thepolypeptide.

The amount of test substance or compound which may be added to an assayof the invention will normally be determined by trial and errordepending upon the type of compound used. Typically, from about 0.01 to100 μM concentrations of putative inhibitor compound may be used, forexample from 0.1 to 10 μM. Greater concentrations may be used when apeptide is the test substance.

Compounds which may be used may be natural or synthetic chemicalcompounds used in drug screening programmes. Extracts of plants whichcontain several characterised or uncharacterised components may also beused. A further class of putative inhibitor compounds can be derivedfrom the STRAP polypeptide or the JMY polypeptide and/or p300polypeptide which to which it binds. Peptide fragments of from 5 to 40amino acids, for example from 6 to 10 amino acids from the region of therelevant polypeptide responsible for interaction, may be tested fortheir ability to disrupt such interaction.

Other candidate inhibitor compounds may be based on modelling the3-dimensional structure of a polypeptide or peptide fragment and usingrational drug design to provide potential inhibitor compounds withparticular molecular shape, size and charge characteristics.

Following identification of a substance which modulates or affectspolypeptide activity, the substance may be investigated further.Furthermore, it may be manufactured and/or used in preparation, i.e.manufacture or formulation, of a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

Thus, the present invention extends in various aspects not only to asubstance identified as a modulator of polypeptide activity, inaccordance with what is disclosed herein, but also a pharmaceuticalcomposition, medicament, drug or other composition comprising such asubstance, a method comprising administration of such a composition to apatient, e.g. for treatment (which may include preventative treatment)of a condition related to stress induced p53 dependent cell apoptosis,such as DNA damage (for example caused by UV radiation), cancer-therapygenotoxicity (for example, caused by chemo- or radiation therapy), p53dependent neuronal death in the central nervous system (i.e. brain orspinal cord injury), preservation of tissues or organs prior totransplant, preparation of host for bone marrow transplant, reducingneuronal damage during seizures and suppression of cell aging, use ofsuch a substance in manufacture of a composition for administration,e.g. for treatment of a condition related to p53 dependent cellapoptosis (such as hyperthermia, hypoxia, stroke, ischemia, acuteinflammation, burn or cell aging), and a method of making apharmaceutical composition comprising admixing such a substance with apharmaceutically acceptable excipient, vehicle or carrier, andoptionally other ingredients.

Cancer therapy includes radio- and chemo-therapy.

A substance identified using as a modulator of polypeptide or promoterfunction may be peptide or non-peptide in nature. Non-peptide “smallmolecules” are often preferred for many in vivo pharmaceutical uses.Accordingly, a mimetic or mimic of the substance (particularly if apeptide) may be designed for pharmaceutical use. The designing ofmimetics to a known pharmaceutically active compound is a known approachto the development of pharmaceuticals based on a “lead” compound. Thismight be desirable where the active compound is difficult or expensiveto synthesise or where it is unsuitable for a particular method ofadministration, e.g. peptides are not well suited as active agents fororal compositions as they tend to be quickly degraded by proteases inthe alimentary canal. Mimetic design, synthesis and testing may be usedto avoid randomly screening large number of molecules for a targetproperty.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g. by substituting each residue in turn. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modelled toaccording its physical properties, e.g. stereochemistry, bonding, sizeand/or charge, using data from a range of sources, e.g. spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conveniently be selected so thatthe mimetic is easy to synthesise, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimisation ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

Mimetics of substances identified as having ability to modulate STRAPpolypeptide activity using a screening method as disclosed herein areincluded within the scope of the present invention. A polypeptide,peptide or substance able to modulate activity of a STRAP polypeptideaccording to the present invention may be provided in a kit, e.g. sealedin a suitable container which protects its contents from the externalenvironment. Such a kit may include instructions for use.

A convenient way of producing a polypeptide according to the presentinvention is to express nucleic acid encoding it, by use of the nucleicacid in an expression system. Accordingly, the present invention alsoencompasses a method of making a polypeptide (as disclosed), the methodincluding expression from nucleic acid encoding the polypeptide(generally nucleic acid according to the invention). This mayconveniently be achieved by growing a host cell in culture, containingsuch a vector, under appropriate conditions which cause or allowexpression of the polypeptide. Polypeptides may also be expressed in invitro systems, such as reticulocyte lysate.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, eukaryotic cells such as mammalian and yeast, and baculovirussystems. Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, COS cells and many others. A common,preferred bacterial host is E. coli. Suitable vectors can be chosen orconstructed, containing appropriate regulatory sequences, includingpromoter sequences, terminator fragments, polyadenylation sequences,enhancer sequences, marker genes and other sequences as appropriate.Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate.For further details see, for example, Molecular Cloning: a LaboratoryManual: 2nd edition, Sambrook et al., 1989, Cold Spring HarborLaboratory Press. Many known techniques and protocols for manipulationof nucleic acid, for example in preparation of nucleic acid constructs,mutagenesis, sequencing, introduction of DNA into cells and geneexpression, and analysis of proteins, are described in detail in CurrentProtocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons,1992.

Thus, a further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. The nucleic acid of theinvention may be integrated into the genome (e.g. chromosome) of thehost cell. Integration may be promoted by inclusion of sequences whichpromote recombination with the genome, in accordance with standardtechniques. The nucleic acid may be on an extra-chromosomal vectorwithin the cell.

A still further aspect provides a method which includes introducing thenucleic acid into a host cell. The introduction, which may (particularlyfor in vitro introduction) be generally referred to without limitationas “transformation”, may employ any available technique. For eukaryoticcells, suitable techniques may include calcium phosphate transfection,DEAE-Dextran, electroporation, liposome-mediated transfection andtransduction using retrovirus or other virus, e.g. vaccinia or, forinsect cells, baculovirus. For bacterial cells, suitable techniques mayinclude calcium chloride transformation, electroporation andtransfection using bacteriophage.

Marker genes such as antibiotic resistance or sensitivity genes may beused in identifying clones containing nucleic acid of interest, as iswell known in the art.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells (which may include cellsactually transformed although more likely the cells will be descendantsof the transformed cells) under conditions for expression of the gene,so that the encoded polypeptide is produced. If the polypeptide isexpressed coupled to an appropriate signal leader peptide it may besecreted from the cell into the culture medium. Following production byexpression, a polypeptide may be isolated and/or purified from the hostcell and/or culture medium, as the case may be, and subsequently used asdesired, e.g. in the formulation of a composition which may include oneor more additional components, such as a pharmaceutical compositionwhich includes one or more pharmaceutically acceptable excipients,vehicles or carriers (e.g. see below).

Introduction of nucleic acid may take place in vivo by way of genetherapy, as discussed below. A host cell containing nucleic acidaccording to the present invention, e.g. as a result of introduction ofthe nucleic acid into the cell or into an ancestor of the cell and/orgenetic alteration of the sequence endogenous to the cell or ancestor(which introduction or alteration may take place in vivo or ex vivo),may be comprised (e.g. in the soma) within an organism which is ananimal, particularly a mammal, which may be human or non-human, such asrabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep,goat, cattle or horse, or which is a bird, such as a chicken.Genetically modified or transgenic animals or birds comprising such acell are also provided as further aspects of the present invention.

In another aspect of the invention, there is provided a method forproducing a transgenic non-human mammal, particularly a rodent such as amouse, by incorporating a lesion into the locus of a STRAP gene.

This may be achieved in a variety of ways. A typical strategy is to usetargeted homologous recombination to replace, modify or delete thewild-type STRAP gene in an embryonic stem (ES) cell. An targeting vectoris introduced into ES cells by electroporation, lipofection ormicroinjection. In a few ES cells, the targeting vector pairs with thecognate chromosomal DNA sequence and transfers the desired mutationcarried by the vector into the genome by homologous recombination.Screening or enrichment procedures are used to identify the transfectedcells, and a transfected cell is cloned and maintained as a purepopulation. Next, the altered ES cells are injected into the blastocystof a preimplantation mouse embryo or alternatively an aggregationchimera is prepared in which the ES cells are placed between twoblastocysts which, with the ES cells, merge to form a single chimericblastocyst. The chimeric blastocyst is surgically transferred into theuterus of a foster mother where the development is allowed to progressto term. The resulting animal will be a chimera of normal and donorcells. Typically the donor cells will be from an animal with a clearlydistinguishable phenotype such as skin colour, so that the chimericprogeny is easily identified. The progeny is then bred and itsdescendants cross-bred, giving rise to heterozygotes and homozygotes forthe targeted mutation. The production of transgenic animals is describedfurther by Capecchi, M, R., 1989, Science 244; 1288-1292; Valancius andSmithies, 1991, Mol. Cell. Biol. 11; 1402-1408; and Hasty et al, 1991,Nature 350; 243-246, the disclosures of which are incorporated herein byreference.

Homologous recombination in gene targeting may be used to replace thewild-type STRAP gene with a specifically defined mutant form (e.gtruncated or containing one or more substitutions).

The invention may also be used to replace the wild-type gene with amodified gene capable of expressing a wild-type or otherwise activeSTRAP polypeptide, where the expression may be selectively blockedeither permanently or temporarily. Permanent blocking may be achieved bysupplying means to delete the gene in response to a signal. An exampleof such a means is the cre-lox system where phage lox sites are providedat either end of the transgene, or at least between a sufficient portionthereof (e.g. in two exons located either side or one or more introns).Expression of a cre recombinase causes excision and circularisation ofthe nuclei acid between the two lox sites. Various lines of transgenicanimals, particularly mice, are currently available in the art whichexpress cre recombinase in a developmentally or tissue restrictedmanner, see for example Tsien, Cell, Vol.87(7): 1317-1326, (1996) andBetz, Current Biology, Vol.6(10): 1307-1316 (1996). These animals may becrossed with lox transgenic animals of the invention to examine thefunction of the STRAP gene. An alternative mechanism of control is tosupply a promoter from a tetracyline resistance gene, tet, to thecontrol regions of the STRAP locus such that addition of tetracyline toa cell binds to the promoter and blocks expression of the STRAP gene.

Transgenic targeting techniques may also be used to delete the STRAPgene. Methods of targeted gene deletion are described by Brenner et al,WO94/21787 (Cell Genesys), the disclosure of which is incorporatedherein by reference.

Homologous recombination may also be used to produce “knock in” animalswhich express a polypeptide of the invention in the form of a fusionprotein, fused to a detectable tag such as β-galactosidase or greenfluorescent protein. Such transgenic non-human mammals may be used inmethods of determining temporal and spatial expression of the STRAP geneby monitoring the expression of the detectable tag.

A further alternative is to target control sequences responsible forexpression of the STRAP gene.

The invention extends to transgenic non-human mammals obtainable by suchmethods and to their progeny. Such mammals may be homozygous orheterozygous. Such mammals include mice, rodents, rabbits, sheep, goats,pigs.

Transgenic non-human mammals may be used for experimental purposes instudying the role of STRAP in regulating the cell cycle and in thedevelopment of therapies designed to target the interaction of STRAPwith other cellular factors, particularly p300 and JMY. By“experimental” it is meant permissible for use in animal experimentationor testing purposes under prevailing legislation applicable to theresearch facility where such experimentation occurs.

Instead of or as well as being used for the production of a polypeptideencoded by a transgene, host cells may be used as a nucleic acid factoryto replicate the nucleic acid of interest in order to generate largeamounts of it. Multiple copies of nucleic acid of interest may be madewithin a cell when coupled to an amplifiable gene such as dihyrofolatereductase (DHFR), as is well known. Host cells transformed with nucleicacid of interest, or which are descended from host cells into whichnucleic acid was introduced, may be cultured under suitable conditions,e.g. in a fermentor, taken from the culture and subjected to processingto purify the nucleic acid. Following purification, the nucleic acid orone or more fragments thereof may be used as desired.

The provision of the novel STRAP polypeptide also enables, for the firsttime, the production of antibodies able to bind specifically to thismolecule.

Accordingly, a further aspect of the present invention provides anantibody able to bind specifically to the polypeptide whose sequence isgiven in a figure herein. Such an antibody may be specific in the senseof being able to distinguish between the polypeptide it is able to bindand other human polypeptides for which it has no or substantially nobinding affinity (e.g. a binding affinity of about 1000× less). Specificantibodies bind an epitope on the molecule which is either not presentor is not accessible on other molecules. Antibodies according to thepresent invention may be specific for the wild-type polypeptide.Antibodies according to the invention may be specific for a particularmutant, variant, allele or derivative polypeptide as between thatmolecule and the wild-type polypeptide, so as to be useful in diagnosticand prognostic methods as discussed below. Antibodies are also useful inpurifying the polypeptide or polypeptides to which they bind, e.g.following production by recombinant expression from encoding nucleicacid.

Preferred antibodies according to the invention are isolated, in thesense of being free from contaminants such as antibodies able to bindother polypeptides and/or free of serum components. Monoclonalantibodies are preferred for some purposes, though polyclonal antibodiesare within the scope of the present invention.

The invention further provides immunological assays which comprise:

-   -   (a) bringing a body sample from said subject into contact, under        binding conditions, with an antibody of the invention; and    -   (b) determining whether said antibody has been able to bind to a        polypeptide in said sample.

Antibodies may be obtained using techniques which are standard in theart. Methods of producing antibodies include immunising a mammal (e.g.,mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and screened,preferably using binding of antibody to antigen of interest. Forinstance, Western blotting techniques or immunoprecipitation may be used(Armitage et al., 1992, Nature 357: 80-82). Isolation of antibodiesand/or antibody-producing cells from an animal may be accompanied by astep of sacrificing the animal.

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047.

Suitable peptides for use in immunising an animal and/or isolatinganti-STRAP antibody include peptides which have the residues 36-46 ofFIG. 1 (SEQ ID NO:1 or SEQ ID NO:2).

Antibodies according to the present invention may be modified in anumber of ways. Indeed, the term “antibody” should be construed ascovering antibody fragments such as Fab and scFv fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimicks that of an antibody enablingit to bind an antigen or epitope.

The reactivities of antibodies on a sample may be determined by anyappropriate means. Tagging with individual reporter molecules is onepossibility. The reporter molecules may directly or indirectly generatedetectable, and preferably measurable, signals. The linkage of reportermolecules may be directly or indirectly, covalently, e.g. via a peptidebond or non-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

One favoured mode is by covalent linkage of each antibody with anindividual fluorochrome, phosphor or laser dye with spectrally isolatedabsorption or emission characteristics. Suitable fluorochromes includefluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

Antibodies according to the present invention may be used in screeningfor the presence of a polypeptide, for example in a test samplecontaining cells or cell lysate as discussed, and may be used inpurifying and/or isolating a polypeptide according to the presentinvention, for instance following production of the polypeptide byexpression from encoding nucleic acid therefor. Antibodies may modulatethe activity of the polypeptide to which they bind and so may be usefulin a therapeutic context (which may include prophylaxis) to modulate thep53 dependent response to cellular stress.

The present invention also provides a substance as described herein foruse in a pharmaceutical composition for the modulation of the p53dependent cell stress response in an individual. Whether it is apolypeptide, antibody, peptide, nucleic acid molecule, small molecule orother pharmaceutically useful compound according to the presentinvention that is to be given to an individual, administration ispreferably in a “prophylactically effective amount” or a“therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may include, in additionto active ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,or Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Targeting therapies may be used to deliver the active agent morespecifically to certain types of cell, by the use of targeting systemssuch as antibody or cell specific ligands. Targeting may be desirablefor a variety of reasons; for example if the agent is unacceptablytoxic, or if it would otherwise require too high a dosage, or if itwould not otherwise be able to enter the target cells.

Instead of administering an agent directly, it may be produced in targetcells by expression from an encoding gene introduced into the cells,e.g. in a viral vector (see below). The vector may be targeted to thespecific cells to be treated, or it may contain regulatory elementswhich are switched on more or less selectively by the target cells.Viral vectors may be targeted using specific binding molecules, such asa sugar, glycolipid or protein such as an antibody or binding fragmentthereof. Nucleic acid may be targeted by means of linkage to a proteinligand (such as an antibody or binding fragment thereof) via polylysine,with the ligand being specific for a receptor present on the surface ofthe target cells.

An agent may be administered in a precursor form, for conversion to anactive form by an activating agent produced in, or targeted to, thecells to be treated.

Pharmaceutical compositions as described herein may be used foranti-sense regulation of gene expression, e.g. targeting an antisensenucleic acid molecule to cells in which a mutant form of the gene isexpressed, the aim being to reduce production of the mutant geneproduct. Other approaches to specific down-regulation of genes are wellknown, including the use of ribozymes designed to cleave specificnucleic acid sequences. Ribozymes are nucleic acid molecules, actuallyRNA, which specifically cleave single-stranded RNA, such as mRNA, atdefined sequences, and their specificity can be engineered. Hammerheadribozymes may be preferred because they recognise base sequences ofabout 11-18 bases in length, and so have greater specificity thanribozymes of the Tetrahymena type which recognise sequences of about 4bases in length, though the latter type of ribozymes are useful incertain circumstances. References on the use of ribozymes includeMarschall, et al. Cellular and Molecular Neurobiology, 1994. 14(5): 523;Hasselhoff, Nature 334: 585 (1988) and Cech, J. Amer. Med. Assn., 260:3030 (1988).

Aspects of the present invention will now be illustrated with referenceto the accompanying figures described below by experimentalexemplification, by way of example and not limitation. Further aspectsand embodiments will be apparent to those of ordinary skill in the art.All documents mentioned in this specification are hereby incorporatedherein by reference.

Experimental

Materials and Methods

Plasmids

The following plasmids were used; pCMV-p300, pCMV-JMY, pVP16-JMY,pG4-p300, pET-JMY, pCMV-HA-JMY469-558, pCMV-HA-JMY1-403,PCMV-HA-JMY119-403, pET-JMY1-504, pCMV-HA-JMY502-983, pCMV-HA-JMY1-119,pCMV-HA-JMY468-558, pCMV-HA-JMY683-983 and pCMV-HA-NAP2 were asdescribed (Shikama et al., 2000; submitted). Flag-tagged p300,pGEXT-2T-p300744-1571, pGEXT-2T-p3001572-2370, pCMV-p300619-1303,pCMV-p3001257-2284, pCMV-p3001257-19 21, pCMV-p53, pCMV-p5322/23, (Linet al., 1994), pBax-luc (Miyashita and Reed, 1995), pWWP-luc (El-Deiryet al., 1993), pCMV-β-gal (Zamanian and La Thangue, 1992), pG5-luc andpTG13 (Lee et al., 1998) and pCMV-HDM2 (Loughran and La Thangue, 2000).

For the preparation of the STRAP expression vectors, pG4-STRAP8-438 wasmade by subcloning the appropriate region of the STRAP cDNA intoEcoRI/NotI sites. pVP16-STRAP8-123 was made by subcloning intoBamHI/XhoI site. pETSTRAP8-438 and pETSTRAP8-123 were constructed bysubcloning into pET28A and pET28C respectively, digested withBamHI/XhoI. pCMV-HA-STRAP8-438, pCMV-HA-STRAP8-205,pCMV-HA-STRAP123-205, pCMV-HA-STRAP206-438, pCMV-HA-STRAP206-327 andpCMV-HA-STRAP328-438 were created by subcloning the appropriate cDNAfragment into the BamHI/XhoI site or for pCMV-HA-STRAP8-123 into theXhoI/XbaI site of pCMV-HA vector.

Antisera.

The following antisera were used; the p53 monoclonal antibody DO-1(Santa Cruz), anti-HA monoclonal antibody Y-11 (Boehringer Mannheim),anti-p300 monoclonal antibody Ab-1 (Calbiochem), anti-p300 rabbitpolyclonal C20 (Santa Cruz), anti-p300 rabbit polyclonal antibody N-15(Santa Cruz) and anti-JMY rabbit polyclonal antibody 789 (Shikama etal., 1999). The STRAP rabbit anti-peptide antisera, 15, was prepared bystandard procedures against a STRAP peptide representing from residue 30to 46.

Isolation of STRAP.

The yeast strain CTY10.5 containing the LexAβ-galactosidase reportervector pLex (His) was as previously described (Buck et al., 1995).pLex-JMY was made by inserting into the EcORI site in frame the fragmentof JMY corresponding to amino acids 469-558 with the DNA binding domainof LexA. Screening a 10.5 day mouse embryo random primed cDNA libraryfused to the VP16 trans-activation domain (Vojtek et al., 1993) yieldeda single positive clone containing 341 bp of the STRAP cDNA sequence.

Full-length STRAP cDNAs were isolated through the combined approach ofscreening cDNA libraries prepared from PCC4 mouse teratocarcinoma cells(Stratagene) and RACE (Clontech) using a testis cDNA library.

Transient Transfections and Reporter Assays.

For transfection into SAOS2 or U2OS cells, cells were incubated in DMEMcontaining 10% serum throughout and transfected with pBax-luc (Miyashitaand Reed, 1995), pWWP-luc (El-Deiry et al., 1993), or pTG13 (Lee et al.,1998) expression vector for p53 (pCMV-p53), together with the indicatedamounts of the STRAP expression vector and harvested 34-36 hpost-transfection (Sørensen et al., 1996; Shikama et al., 1999). Alltransfections were performed using the calcium phosphate procedure andincluded the internal control pCMV-β-gal (Zamanian and La Thangue,1992). The mammalian two-hybrid assay was performed in U2OS cells asdescribed using the Gal4-responsive reporter pG5-luc andpG4-p300611-2283 (Lee et al., 1998) together with pVP16-STRAP, orpG4-STRAP hybrid together with pVP16-JMY.

Immunoprecipitation and Immunoblotting.

For immunoprecipitation, p53−/− MEFs, U2OS and SAOS2 cells weretransfected with the expression vectors pCMV-p300 and pCMV-JMY andpCMV-HA-STRAP. After 48 h, cells were harvested in TNN buffer (50 mMTris-HCl (pH7.4), 5 mM EDTA, 0.5% NP40, 50 mM NaF, 1 mM DTT, 0.2 mMsodium orthovanadate) and protease inhibitor cocktail (1 mM PMSF,leupeptin, aprotinin and pepstatin (1 μg/ml)) containing 120 mM NaCl andincubated on ice for 30 min.

The cell extract was first precleared for 1 h with protein G and thenimmunoprecipitated with the anti-HA monoclonal antibody Y11, which wascollected with protein-A agarose. The agarose beads were collected andwashed three times in the extraction buffer before denaturation andSDS-PAGE. Immunoblotting was subsequently performed with either ananti-p300 monoclonal antibody Ab-1 or anti-JMY polyclonal antibody.

For p53 stability studies p53−/− MEF, were transfected as described withpCMV-p53, pCMV-HDM2 and the indicated amounts of pCMV-HA-STRAP. 6 hafter transfection the cells were harvested as described and submittedto SDS-PAGE and immunoblot. In the case of U2OS cells, endogenous p53protein was followed after the transfection of pCMV-HA-STRAP. Cells wereharvested and the cell extracts prepared as described above. The p53protein was detected by immunoblotting with DO-1.

Biochemical Binding Assay.

Flag-tagged full-length p300 protein was expressed in the baculovirusexpression system and bound to M2-anti-flag antibody agarose (Kodak).For the control beads, the M2-anti-flag antibody agarose was treatedwith cell extract from uninfected Sf9 cells.

The various JMY and STRAP proteins were in vitro translated by usingTNT-coupled reticulocyte lysate system (Promega) in the presence of[35S]Met/Cys (Amersham). Each translation reaction (25 μl) was incubatedeither with the beads coupled to the flag-tagged p300 or the controlbeads for 2 h at 4° C. The protein complex on the beads was washed threetimes in TNN buffer containing 100 mM NaCl and eluted in 2×SDS samplebuffer and loaded on to an SDS-PAGE gel. The proteins were detected byautoradiography.

For JMY469-558, in vitro translation was carried out in the absence ofradioactive amino acids. The protein was detected by immunoblottingusing anti-HA Y11 monoclonal antibody. Wild-type STRAP in pET28a(Novagen) was expressed and purified through His-tag chromatographyaccording to the manufacturer's instructions (Pharmacia).

The eluted fraction containing His-STRAP was dialysed against 50 mM Tris(pH 7.5), 100 mM KCl, 20% glycerol, 0.2 mM DTT, 0.2 mM PMSF. For the invitro binding assay, flag-tagged full-length p300 was expressed in thebaculovirus system and bound to M2-anti-flag antibody agarose (Kodak).For control beads, the M2-anti-flag antibody agarose was treated withcell extract from uninfected Sf9 cells. Wild-type JMY and STRAP inpET28a vector (Novagen) were expressed in bacteria and purified throughnickel chromatography according to the manufacturer's instructions(Pharmacia). The eluted fraction containing His-STRAP and His-JMYprotein was dialysed against 50 mM Tris (pH 7.5), 100 mM KCl, 20%glycerol, 0.2 mM DTT, 0.2 mM PMSF.

The binding assay using flag-p300 was performed for 3 h at 4° C. in abuffer containing Tris-pH 7.5, 250 mM NaCl, 0.1% NP40, 10% glycerol, 1.5mM MgCl₂, 0.2 mM EDTA, 0.5 mg/ml BSA, 0.5 mM DTT, 0.5 mM PMSF andprotease inhibitors. After binding, the beads were washed three times in50 mM Tris-pH 7.5, 250 mM NaCl, 0.1% NP40, 10% glycerol, 1.5 mM MgCl2,0.2 mM EDTA, 0.1 mM DTT, 0.1 mM PMSF and protease inhibitors. The levelof JMY bound to p300 was detected by immunoblotting with the anti-JMYantiserum.

Cyclohexamide Treatment.

U2OS cells were transfected with the pCMV-HA-STRAP, pCMV-HA-JMY,pCMV-p300 and for control empty pcDNA3 expression vectors. After 48 h oftransfection, cyclohexamide (10 μg/ml) was added to the cells for theindicated time periods and cells were harvested in TNN buffer (Maki andHowley, 1997). The cell extract was loaded on SDS-PAGE and the p53protein was detected on nitrocellulose membrane with anti-p53 monoclonalantibody DO1.

Etoposide Treatment

Cells were transfected with the pCMV-HA-STRAP, pCMV-JMY or pCMV-p53 andfor the control empty pcDNA3 expression vector. Etoposide at a finalconcentration of 200 nM and 400 nM was added to the cells 12 h beforeharvesting. Cells were harvested in TNN buffer and submitted to eitherimmunoblotting or luciferase activity assays.

Flow Cytometry.

SAOS2 cells were transfected with pCMV-p53 or pCMV-p5322/23 (5 □g)either alone or together with pCMV-STPAP. Flow cytometry was performedas described previously (de la Luna et al. 1999; Shikama et al., 1999).

Irradiation.

Two hours after irradiation (10GyIR and 80J/m2 UV) A31, p53−/− andp53−/−/MDM2−/− MEFs, were washed twice with ice cold PBS (pH7.4). Lysiswas performed on ice for 20 min in lysis buffer TNN (Tris HCl pH7.5, 50mM, NaCl 120 mM, EDTA 1 mM, 0.5% Nonidet P-40, 1 mM PMSF, proteaseinhibitors). Lysates were centrifuged for 20 min, 12,000 rpm, at 4° C.and equal amounts of protein were loaded onto an SDS gel and the amountof STRAP1 determined after immunoblotting with the anti-STRAP antibody.

Results

Isolation and Characterization of STRAP.

A yeast two-hybrid assay was used to screen for proteins that arecapable of physically interacting with the JMY co-factor, which is knownto form a complex with p300 (Shikama et al., 1999). For the bait, weused a hybrid protein in which an internal domain of JMY from residue469 to 558, containing a region previously assigned to the interactionwith p300 (Shikama et al., 1999), was fused to the LexA DNA bindingdomain.

Screening of a 10.5 d.p.c. mouse embryo activation domain-tagged libraryidentified a partial cDNA clone of novel sequence. Subsequent isolationand sequence analysis of the full-length cDNA indicated that it encodeda protein of 440 residues which lacks significant homology to any otherknown protein or nucleotide sequence on the currently available databases (FIG. 1). Because of the properties of this new protein inperforming a key role in facilitating stress-responsive protein-proteininteractions within the p300 co-activator complex, the protein has beendesignated STRAP, to reflect its function as a stress-responsiveactivator of p300.

A remarkable feature of STRAP is the presence of six tetratricopeptiderepeat (TPR) motifs that are distributed throughout the entire length ofthe protein (FIGS. 4 and 5). Whilst a variety of other proteins havebeen found to possess TPR motifs (Lamb et al., 1995; Blatch and Lassle,1999), STRAP appears to be quite unusual in its tandem distribution ofTPR motifs. Based on multiple sequence alignments, there appear to be nostrictly conserved residues in the 34 amino acid residue TPR motif(Blatch and Lassle, 1999). There are however, strong preferences forsmall hydrophobic residues at certain positions. The TPR motifs in STRAPfit in well with the existing information on the composition of TPRmotifs.

An analysis of the pattern of expression of STRAP by northern blottingwith RNA prepared from different mouse tissues including heart, brain,spleen, lung, liver, kidney and testis, indicated that expression iswidespread, similar results being obtained in cell lines derived fromdiverse origins.

STRAP Interacts with Distinct Components of the p300 Co-activatorComplex.

The TPR motif is a helical motif that can function in protein-proteininteractions (Lamb et al., 1995; Blatch and Lassle, 1999). Since STRAPwas isolated in a yeast two-hybrid screen using a domain of JMY as thebait, we assessed if STRAP could bind to JMY in mammalian cells andthereafter studied its interaction with other components of theco-activator complex.

We performed a series of two-hybrid assays in U2OS cells, in which wefound that VP16-JMY could induce the activity of a hybrid protein inwhich the STRAP sequence was fused to the Gal4 DNA binding domain,referred to as G4-STRAP, but not G4 alone (FIG. 5). Taking a similarapproach, VP16-STRAP could induce G4-p300 (FIG. 6). These resultsprovide indication that STRAP interacts with at least two components ofthe p300 co-activator complex, namely JMY and p300.

U2OS cells were transfected with expression vectors for HA-tagged STRAPtogether with JMY or p300, and assessed their interaction byimmunoprecipitation with an anti-HA monoclonal antibody followed byimmunoblotting with either anti-JMY or anti-p300. In both experiments,STRAP specifically co-immunoprecipitated with JMY and p300.

An anti-peptide antibody against STRAP was prepared to determine whethersimilar interactions occur under physiological conditions. This antibodyreacted specifically with bacterially expressed wild-type STRAP, andrecognised the exogenous STRAP polypeptide, of about 60,000 molecularweight, in transfected cells. Moreover, an endogenous polypeptide ofsimilar molecular weight was recognised by the anti-STRAP antibody inmurine A31 cells. Binding of the antibody to the 60,000 molecular weightSTRAP polypeptide was blocked upon the presence of the homologouspeptide. We used this anti-STRAP antibody to test whether p300 and STRAPform a complex under physiological conditions. In anti-p300immunoprecipitates from murine A31 cells, we found STRAP to be presentin the p300 immunocomplex. These results provide strong indication thatSTRAP and p300 exist as a complex under normal physiological conditions.

Binding Domains in STRAP, p300 and JMY.

The binding domains in STRAP for JMY and p300 were determined through abiochemical assay in which different regions of STRAP were in vitrotranslated and thereafter assessed for binding to either his-taggedwild-type JMY or flag-tagged wild-type p300.

Out of a panel of STRAP derivatives, two distinct regions were found tobe responsible for the interaction with JMY, one located in theN-terminal region up to residue 123, and the other within residue 123 to205; the C-terminal half of STRAP (from residue 206) exhibited littlebinding activity for JMY.

In a continuation of this analysis, we studied the interaction domain inSTRAP for p300. An analysis of the same set of STRAP mutant derivativesindicated that the predominant p300 interaction domain was, in contrast,localised in the C-terminal half of STRAP, and encompassed from residue206 to 438. Overall, these binding studies established that STRAPpossesses two separable and distinct regions of interaction for JMY andp300, the binding domain for JMY being primarily localised within theN-terminal half with the p300 interaction domain being present in theC-terminal half (FIG. 7).

A similar approach was taken to elucidate the domains in JMY and p300that are responsible for interacting with STRAP. An analysis of thebinding properties of a panel of in vitro translated JMY derivativesindicated that JMY harbours at least two interaction domains for STRAP,one of which resides within the N-terminal 119 residues, the other beingbroadly defined to the C-terminal region from residue 683 to 983. Inaddition, another STRAP binding domain in JMY mapped to residue 468 to558, as this region was used in the two-hybrid screen to isolate STRAP.Thus, JMY contains at least three distinct interaction domains for STRAP(FIG. 7).

Finally, we investigated a panel of p300 derivatives for their STRAPbinding activity. As expected, wild-type p300 bound to STRAP, andfurther analysis of the binding properties of the mutant derivativesmapped two p300 interaction domains, one to the N-terminal 595 residues,with the other one located in the C-terminal region between residues1572 to 1921. Taken together, this analysis of the binding properties ofSTRAP, JMY and p300 established that STRAP can bind specifically to JMYand p300, and that it does so through distinct domains within the N- andC-terminal regions of the protein (see FIG. 7). Similarly, JMY and p300possess dedicated domains that allow each protein to interact with STRAPand, as previously documented, with each other.

STRAP Facilitates the Interaction Between JMY and p300.

The level of JMY that co-immunoprecipitated with p300 from cellstransfected with JMY and p300 was determined, and thereafter the effectof co-expressing STRAP. At the same time, we investigated the level ofJMY, p300 and STRAP in transfected cells which indicated thatco-expressing STRAP, p300 and JMY caused an accumulation of JMY andSTRAP. In the same cell extract we found, as expected (Shikama et al.,1999), that JMY co-immunoprecipitated with p300 and, importantly, thatthere was a highly significant increase in the level of JMY in the p300immunocomplex in the presence of STRAP. Moreover, since the amount ofp300 in the immunocomplex was similar in the absence or presence ofSTRAP, the increased level of co-immunoprecipitated JMY results from theinfluence of STRAP on the recruitment of JMY into the p300 complex.

A mammalian two-hybrid assay was used to investigate whether STRAPaffected the interaction between JMY and p300. As in previous studies,in a two-hybrid assay G4-p300 and VP16-JMY could interact with eachother (Shikama et al., 1999). Under these conditions, STRAP caused asignificant increase in activity. A titration of STRAP indicated thatfurther increases in the amount of co-transfected STRAP caused areduction in the two-hybrid signal (FIG. 8). A possible explanation forthis phenomenon is that there is an optimal level of STRAP that favoursthe interaction between JMY and p300, above which the level of STRAPout-titrates the amount of p300 and JMY, and rather than recruiting p300and JMY into a ternary complex, STRAP binds to each as heterodimer andthus interferes with the two-hybrid interaction.

We then purified recombinant p300, JMY and STRAP and studied theinfluence of STRAP upon the interaction between JMY and p300 in abiochemical assay. Previous studies have established that JMY and p300can form a protein complex, and identified the domains in each proteinthat are responsible for this interaction (Shikama et al., 1999). Underconditions where purified recombinant p300 and JMY could weakly bind toeach other, the addition of STRAP increased the efficiency of theirinteraction, enhancing the amount of JMY that bound to p300. Thespecificity of this effect was established through a variety of controltreatments, including the effect of a STRAP mutant derivative containingthe N-terminal region (residue 8 to 123), that harbours one complete TPRmotif, and is capable of binding to JMY, but not p300 (FIG. 7). Theaddition of a similar amount of STRAP8-123 failed to affect theinteraction between. p300 and JMY, thus establishing the specific effectof STRAP on the JMY/p300 interaction.

STRAP Augments the p53 Response.

It is known that p300/CBP proteins participate in the control ofp53-dependent transcription, and that during the p53 response alteredp53 stability serves as a major level of regulation through a processwhich perhaps involves p300 (Ko and Prives, 1997; Levine et al., 1997;Shikama et al., 1997). We introduced STRAP into U2OS cells and studiedthe level of endogenous p53 by immunoblotting. Increasing the levels ofSTRAP caused a concomitant increase in p53. Similarly, upon theintroduction of exogenous p53 into p53−/− MEFs, we observed asignificant increase in p53 levels. Mutant derivatives of STRAPrepresenting both N-and C-terminal deletions failed to cause anyalteration in p53 level. We introduced STRAP into U2OS cells to test forany effect on the half-life of p53 and studied endogenous p53 aftertreating the cells with cyclohexamide. We found that endogenous p53 hada half-life of about 30 minutes which, upon the expression of STRAP, wassignificantly lengthened to about 240 minutes. In contrast, theexpression of neither JMY nor p300 had a significant impact on p53half-life, which remained at approximately 30 minutes. Thus, STRAPinduces the p53 protein, which it does so in part by altering the rateof p53 turnover and increasing p53 half-life.

Previous studies have established that the physical interaction of MDM2with the activation domain of p53 abrogates the p53 response bytargeting p53 for degradation (Haupt et al., 1997; Kubbutat et al.,1997). Thus, the level of exogenous p53 in p53−/− MEFs was reduced uponthe co-expression of human MDM2 (referred to as hDM2). Under theseconditions, the presence of STRAP significantly increased the level ofp53, providing indication that STRAP can override the down-regulation ofp53 activity by hDM2, a result that is consistent with the ability ofSTRAP to extend p53 half-life and augment the levels of the p300co-activator complex. It is important to note that under theseexperimental conditions we failed to detect any direct interactionbetween p53 and STRAP.

As p300/CBP and JMY are involved in regulating p53 transcriptionalactivity, we investigated the effect of STRAP on differentp53-responsive promoters. Consistent with its presence in the p300co-activator complex and the ability to extend p53 half-life, we foundSTRAP to be capable of inducing p53 activity on a variety of differentp53-responsive promoters, including waf1, bax and the artificialp53-responsive TG13 promoter (FIG. 9).

We then investigated whether the ability of STRAP to increase p53protein levels and augment transcription correlated with thephysiological properties of p53, namely the ability to promote cellcycle arrest and apoptosis (Levine, 1997). In SAOS2 (p53−/−) tumourcells we identified conditions where the introduction of wild-type p53caused a low but significant increase in the population of apoptosingcells (FIG. 10). Under these conditions, co-expressing STRAP with p53resulted in a marked increase in the size of the apoptosing cellpopulation, in contrast to the effects of STRAP in the absence of p53,which were minimal. These results establish that STRAP augments theapoptotic activity of p53.

The presence of STRAP in the p300 co-activator complex providedindication that STRAP can augment p53-dependent apoptosis throughstimulating p53 transcriptional activity. We assessed whether STRAPcould induce p5322/23, which is a mutant p53 derivative that has tworesidues altered in the activation domain, and which is severelycompromised in transcriptional activity (Lin et al., 1994). Incomparison to wild-type p53, p5322/23 was less efficient at inducingapoptosis but still retained a significant level of activity (FIG. 11).However, in contrast to wild-type p53, the apoptotic activity ofp5322/23 was not affected by co-expressing STRAP, a result that isconsistent with a role for STRAP in stimulating p53 transcription.

Overall, these studies provide indication that STRAP as a component ofthe p300 co-activator complex induces p53 activity in part by increasingthe stability of the p53 protein, and further that this effect directlyinfluences p53-dependent transcription and therefore the p53 response.

STRAP is a Damage-responsive Component of the p300 Co-activator Complex.

We investigated whether the protein level was altered in stressed cellsby studying the effect of etoposide, which is an agent that canefficiently induce the p53 response (Kaufmann, 1998; Arriola et al.,1999). We compared in U2OS cells the induction of STRAP to p53, as wellas to the induction of JMY and NAP (nucleosomal assembly protein), thelatter serving as a control protein that was un likely to be affected bystress. STRAP protein levels increased in response to etoposide,although not as dramatically as observed for p53; in contrast, JMY andNAP failed to undergo a similar response, with NAP levels declining(FIG. 12).

We then investigated the p300/JMY complex in etoposide-treated U2OScells, in the absence and presence of STRAP. The expression of STRAPcaused an increase in the level of JMY bound to p300, which showed afurther and significant increase upon treating the cells with etoposide.Using the same cell extracts, a marked increase in the level ofco-immunoprecipitated JMY with p300 was observed when STRAP expressingcells were treated with etoposide (usually about 10-fold in contrast to2-fold induction of protein level). These results provide indicationthat STRAP is a stress responsive protein that augments the interactionbetween p300 and JMY.

The role of STRAP in regulating the stress-responsive interactionbetween p300 and JMY was further characterised in a two-hybrid assayusing G4-p300 and VP16-JMY. We found that treating U2OS cells withetoposide (200 nM) caused a marked reduction in the level of two-hybridsignal, and that the expression of STRAP completely restored two-hybridactivity (FIG. 13). This effect required the integrity of the STRAPprotein, as a mutant derivative that failed to interact with p300 butretained the ability to bind to JMY, STRAP8-205, could not overcome theeffect of etoposide upon the two-hybrid interaction between G4-p300 andVP16-JMY.

We extended these studies to an analysis of the effect of STRAP upon theetoposide-dependent regulation of wild-type p53 transcriptionalactivity. In a similar fashion to the etoposide effect on the p300/JMYinteraction (FIG. 13), at a high concentration of etoposide, a reductionin p53 activity occurred (FIG. 14). Co-expression of STRAP partiallyovercame the etoposide-dependent down-regulation of p53 transcriptionalactivity and furthermore, the effect of STRAP was dependent upon theintegrity of the wild-type protein, since the expression of STRAP8-123interfered with wild-type STRAP activity, most probably through adominant-negative type of effect (FIG. 14).

These results provide indication that STRAP plays a direct role infacilitating the p53 response in conditions of cellular stress. A fargreater association was observed between STRAP and the p53 transcriptioncomplex in etoposide-treated U2OS cells compared to untreated cells,indicating that the association between STRAP and p300, and thereafterp53, is stress-responsive. These results demonstrate that STRAPfunctions in regulating and maintaining p300 co-activator functionduring cellular stress, and that this role of STRAP contributes to p53activity.

Endogenous STRAP is a Stress-responsive Protein.

The stress responsiveness of endogenous STRAP was determined stressusing the anti-STRAP peptide antibody by studying. STRAP levels in A31cells after treatment with etoposide, as well as other stress-inducingagents. STRAP was found to be effectively induced by etoposide whereasthe effect of ultra-violet light and ionizing radiation was less marked.

The importance of p53 and MDM2 in the stress-regulation of endogenousSTRAP was assessed by monitoring STRAP levels in early passage p53−/− orp53−/−/mdm2−/− mouse embryo fibroblasts (MEFs). STRAP was induced inp53−/− MEFs to a similar extent as that observed in murine A31 cells. Incontrast, little change in STRAP level was observed in the doubleknockout p53−/−/mdm2−/− cells. These results show that STRAP can beinduced independently of p53, but indicate that MDM2 can influence thestress-responsive induction of STRAP.

The stress response control of STRAP described herein allows a number ofimportant conclusions to be made. Firstly, STRAP is a stress-responsiveprotein, since STRAP levels increased in stressed cells. Secondly, theeffect and interaction of STRAP with the p53 associated p300 complexprovide strong indication that STRAP is a functionally importantcomponent of the p53 response. The latter conclusion, in combinationwith the earlier results on the ability of STRAP to foster theinteraction between p300 and JMY, show that a primary function of STRAPis to augment p300 co-activator activity in conditions of the p53response.

STRAP is a Novel TPR Motif Protein.

The characterisation of STRAP has shown that the protein possesses arather unusual domain organisation, since it is composed almost entirelyof a tandem series of TPR motifs (Lamb et al., 1995). The TPR motif hasbeen found in a wide variety of proteins, from prokaryotes toeukaryotes, and is generally believed to represent an ancientprotein-protein interaction module (Blatch and Lassle, 1999). Proteinsthat contain TPR motifs function in diverse physiological processes,including the cell cycle, transcription and the stress response, wherethey usually occur as integral components of multiprotein complexes.

The characteristics ascribed to STRAP as a functionally importantcomponent involved in regulating the p300/CBP co-activator complex fitvery well with the generic properties previously assigned to TPRmotif-containing proteins in regulating and mediating the assembly ofmacromolecular protein complexes. Particularly relevant is theconsiderable body of evidence that highlights the multicomponent natureof p300/CBP co-activator complex, in which STRAP plays a role infacilitating the interaction between p300 and JMY (Shikama et al.,1999). Another notable feature is the fact that STRAP has distinct andseparable TPR motif-containing domains that allow it to bind to proteincomponents of the p300 co-activator complex. Moreover, our results implythat this ability of STRAP to interact with p300 and JMY facilitatestheir interaction, suggesting that STRAP may play a role in maintainingthe functional integrity of the p300/CBP co-activator complex.

STRAP May Facilitate the Assembly of the p300/CBP Co-activator Complex.

It is interesting to speculate on the role of STRAP in the p300/CBPco-activator complex. In this respect, it is noteworthy that althoughproteins with multiple TPR motifs are found in other protein complexes,the TPR motif usually co-exists with domains dedicated to otherfunctions, such as phosphatase activity in the case of PP5 (Ollendorfand Donoghue, 1997). STRAP is unusual in this respect, as the majorityof the protein exists as a series of tandem TPR motifs.

STRAP can interact specifically with JMY and p300, using domains whichat the biochemical level appear to be non-overlapping. Previous studiesindicated that JMY and p300 can directly and specifically bind to eachother (Shikama et al., 1999), an interaction that STRAP enhances througha process that involves the formation of a ternary complex betweenSTRAP, p300 and JMY. STRAP may play a role in regulating the assembly ofthe p300 complex, and represents a key regulatory component incontrolling p300 co-activator activity.

STRAP is Stress-responsive and Augments the p53 Response.

STRAP induces p53-dependent transcription and facilitates the p53response (FIG. 15). Given the documented role of the p300 co-activatorcomplex in regulating p53 activity (Gu et al., 1997; Lill et al., 1997;Lee et al., 1998), this activity may result from the ability of STRAP tofoster the assembly of the p300 co-activator complex during cellularstress. In its regulation of p53, an important property was seen in thelevel of the STRAP protein, which undergoes a stress-responsiveaccumulation when cells were treated with agents such as etoposide,which is known to activate the p53 response by causing double strandedbreaks in genomic DNA (Kaufman, 1998). Thus, our study providesindication that there are a number of steps through which STRAPinfluences the p53 response, whereby the initiating event, namely theSTRAP facilitated assembly of the p300 co-activator complex contributesto a variety of downstream consequences, including an extended p53half-life, in part through modulating MDM2 activity, and thereafter theactivation of p53 target genes that execute the p53 response.

A New Level of Control in the p53 Response.

This document describes the first transcription co-factor that has adedicated role in regulating the assembly of a transcriptionalco-activator complex and which, furthermore, possesses molecularproperties which allow it to function during cellular stress. Theproperties of STRAP are well-suited to this role. STRAP favours andstrengthens complex formation between p300 and JMY, and undergoesstress-responsive protein accumulation. These properties endow STRAPwith the ability to maintain p300 co-activator activity in adversecellular conditions. The properties of STRAP may have an importantimpact on facilitating the cellular stress response which, in the caseof the p53 response, relies upon the transcriptional activation of a setof targets genes that act as a major driving force in delivering andexecuting the response mechanism.

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1. An isolated polypeptide which includes the amino acid sequence shownin SEQ ID NO:1 or the amino acid sequence shown in SEQ ID NO:2.
 2. Apeptide which binds JMY and/or p300 and is capable of modulating p53activity, said peptide being a fragment of an isolated polypeptide whichincludes the amino acid sequence shown in SEQ ID NO:1 or the amino acidsequence shown in SEQ ID NO:2.
 3. An isolated nucleic acid moleculeencoding the polypeptide as shown in SEQ ID NO:2.
 4. An expressionvector comprising the nucleic acid according to claim 3 operably linkedto a regulatory sequence.
 5. An isolated host cell transformed with theexpression vector of claim
 4. 6. A pharmaceutical composition comprisinga polypeptide or peptide fragment according to claim 1 or 2 and apharmaceutically acceptable excipient or carrier.
 7. A method of makinga polypeptide which includes the amino acid sequence shown in SEQ IDNO:2 comprising culturing a host cell transformed with an expressionvector, said vector comprising a nucleic acid molecule encoding apolypeptide shown in SEQ ID NO:2, said nucleic acid being operablylinked to a regulatory sequence, said culturing comprising conditionsfor expression of said polypeptide.
 8. The method of making apolypeptide according to claim 7, comprising testing for binding for JMYor p300.
 9. The method according to claim 7, further comprisingisolating and/or purifying said polypeptide.
 10. The method according toclaim 9 wherein the isolated or purified polypeptide is formulated intoa composition comprising one or more additional components.
 11. An assaymethod for obtaining an agent able to interact with a polypeptide orfragment according to claim 1 or claim 2, including: (i) bringing intocontact said polypeptide or fragment and a putative binding molecule orother test substance; and (ii) determining interaction or bindingbetween the polypeptide or fragment and the test substance.